benzyloxycarbonylleucyl-leucyl-leucine-aldehyde and Neoplasms

Human DNA topoisomerase II is an important target in anticancer therapy. Despite the clinical success of drugs that target topoisomerase II, the development of resistant cancer cells can limit their clinical efficacy. To maximize the therapeutic potential of anticancer drugs, combination therapies and multitarget drugs have been suggested in many studies, where the use of multitarget drugs is advantageous from a pharmacokinetic point of view. There are various different options for the preparation of dual-target or multiple-target inhibitors, as topoisomerase II is both structurally (e.g., topoisomerase I, Hsp90, and kinases) and functionally (e.g., histone deacetylases and proteasome) connected to many validated anticancer targets. In this Perspective, we discuss the scientific background behind targeting topoisomerase II together with a number of other targets important in cancer therapy, review the present status, and discuss further options in the field.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; DNA Topoisomerases, Type II; Drug Design; HSP90 Heat-Shock Proteins; Humans; Neoplasms; Topoisomerase II Inhibitors; Tubulin Modulators

The balance between cell proliferation and apoptosis is critical for normal development and for the maintenance of homeostasis in adult organisms. Disruption of this balance has been implicated in a large number of disease processes, ranging from autoimmunity and neurodegenerative disorders to cancer. The ubiquitin-proteasome pathway, responsible for mediating the majority of intracellular proteolysis, plays a crucial role in the regulation of many normal cellular processes, including the cell cycle, differentiation and apoptosis. Apoptosis in cancer cells is closely connected with the activity of ubiquitin-proteasome pathway. The peptide-aldehyde proteasome inhibitor MG132 (carbobenzoxyl-L-leucyl-L-leucyl-L-leucine) induces the apoptosis of cells by a different intermediary pathway. Although the pathway of induction of apoptosis is different, it plays a crucial role in anti-tumor treatment. There are many cancer-related molecules in which the protein levels present in cells are regulated by a proteasomal pathway; for example, tumor inhibitors (P53, E2A, c-Myc, c-Jun, c-Fos), transcription factors (transcription factor nuclear factor-kappa B, IκBα, HIFI, YYI, ICER), cell cycle proteins (cyclin A and B, P27, P21, IAP1/3), MG132 induces cell apoptosis through formation of reactive oxygen species or the upregulation and downregulation of these factors, which is ultimately dependent upon the activation of the caspase family of cysteine proteases. In this article we review the mechanism of the induction of apoptosis in order to provide information required for research.

Topics: Adult; Apoptosis; Humans; Leupeptins; Neoplasms; Proteasome Endopeptidase Complex; Proteasome Inhibitors

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Humans; Idarubicin; Leupeptins; Neoplasms; Neoplastic Stem Cells

This report reviews the current status of extensive efforts directed towards the interpretation of crosstalk between apoptosis and proteasome to understanding the molecular mechanism of anticancer agents targeting proteasome, with particular focus on MG132 and PS-341. The discovery that all cancer cells have retained the apoptotic death program has offered to the researchers new biochemical targets to design anticancer drugs. Moreover, the demonstration that proteasome inhibition induces apoptosis and sensitizes cancer cells to traditional tumoricidal agents has proposed the proteasome as an attractive target for development of new anticancer drugs. Since then, a number of both naturally occurring and synthetic inhibitors of the proteasome have been identified. The best characterized and most widely used inhibitors of the proteasome are the peptide aldehydes; among these MG132, due to its broad spectrum of action, low cost and rapid reversibility of action, still remains the first choice to study proteasome function in cell and tissue cultures. Recently, a very potent new class of selective and reversible proteasome inhibitors which contains an inhibitory boronate group has been described. PS-341 represent the first of this promising class of agents that could have application in cancer therapy and it is the only that has progressed to clinical trials.

Topics: Animals; Antineoplastic Agents; Apoptosis; Boronic Acids; Bortezomib; Cell Line, Tumor; Clinical Trials as Topic; Cysteine Endopeptidases; Humans; Leupeptins; Models, Biological; Multienzyme Complexes; Neoplasms; Peptide Hydrolases; Phenotype; Protease Inhibitors; Proteasome Endopeptidase Complex; Pyrazines

The involvement of mammalian target of rapamycin (mTOR) in the positive regulation of oncogenesis has been well documented and thus mTOR has emerged as an attractive cancer therapeutic target. Although rapamycin and its analogues (rapalogs) are FDA-approved for the treatment of certain cancers, major success in targeting mTOR, particularly with new generation mTOR kinase inhibitors, for the effective treatment of cancers has not been achieved. Hence, a thorough understanding of the biology of the mTOR axis in cancer is still needed. It is now recognized that programmed death-ligand 1 (PD-L1) expression on cancer cells is a critical mechanism contributing to immunosuppression and immune escape via interacting with program death-1 (PD-1) on immune cells. This study has revealed a previously undiscovered role of the mTOR complex 1 (mTORC1)/p70 S6 kinase (p70S6K) in the negative regulation of PD-L1 on cancer cells and tissues. We demonstrate that disruption of this signaling pathway with mTOR inhibitors, raptor knockdown or p70S6K inhibitors elevated PD-L1 levels in some lung and other cancer cell lines. Elevation of PD-L1 by inhibition of mTORC1/p70S6K signaling is likely due to suppression of β-TrCP-mediated proteasomal degradation of PD-L1, because inhibition of either mTORC1 or p70S6K facilitated β-TrCP degradation accompanied with enhanced PD-L1 protein stabilization. Our current findings indicate the complexity of the mTOR axis in cancer, which should be considered when targeting this axis for effective cancer treatment. Our findings also suggest a strong scientific rationale for enhancing PD-1/PD-L1-targeted cancer immunotherapy through co-targeting mTORC1/p70S6K signaling.

Topics: A549 Cells; B7-H1 Antigen; Benzoxazoles; beta-Transducin Repeat-Containing Proteins; Cycloheximide; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; HCT116 Cells; HEK293 Cells; Humans; Leupeptins; MCF-7 Cells; Mechanistic Target of Rapamycin Complex 1; Morpholines; Naphthyridines; Neoplasms; Protein Stability; Pyrimidines; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; Tumor Cells, Cultured

β-catenin is a major transcriptional activator of the canonical Wnt/β-catenin signaling pathway. It is important for a series of biological processes including tissue homeostasis, and embryonic development and is involved in various human diseases. Elevated oncogenic activity of β-catenin is frequently observed in cancers, which contributes to survival, metastasis and chemo-resistance of cancer cells. However, the mechanism of β-catenin overexpression in cancers is not well defined. Here we demonstrate that the deubiquitination enzyme USP20 is a new regulator of the Wnt/β-catenin signaling pathway. Mechanistically, USP20 regulates the deubiquitination of β-catenin to control its stability, thereby inducing proliferation, invasion and migration of cancer cells. High expression of USP20 correlates with increased β-catenin protein level in multiple cancer cell lines and patient samples. Moreover, knockdown of USP20 increases β-catenin polyubiquitination, which enhances β-catenin turnover and cell sensitivity to chemotherapy. Collectively, our results establish the USP20-β-catenin axis as a critical regulatory mechanism of canonical Wnt/β-catenin signaling pathway with an important role in tumorigenesis and chemo response in human cancers.

Topics: Animals; Antineoplastic Agents; beta Catenin; Cell Line, Tumor; Cell Movement; Cell Survival; Cell Transformation, Neoplastic; Drug Resistance, Neoplasm; Female; HEK293 Cells; Humans; Leupeptins; Mice; Mice, Nude; Neoplasms; RNA Interference; RNA, Small Interfering; Ubiquitin Thiolesterase; Ubiquitination; Wnt Signaling Pathway

Receptor-interacting protein kinase 3 (RIPK3) is a serine/threonine kinase with essential function in necroptosis. The activity of RIPK3 is controlled by phosphorylation. Once activated, RIPK3 phosphorylates and activates the downstream effector mixed lineage kinase domain-like (MLKL) to induce necroptosis. In certain situations, RIPK3 has also been shown to promote apoptosis or cytokine expression in a necroptosis and kinase-independent manner. The ubiquitin-proteasome system is the major pathway for selective degradation of cellular proteins and thus has a critical role in many cellular processes such as cell survival and cell death. Clinically, proteasome inhibition has shown promise as an anti-cancer agent. Here we show that the proteasome inhibitors MG132 and bortezomib activate the RIPK3-MLKL necroptotic pathway in mouse fibroblasts as well as human leukemia cells. Unlike necroptosis induced by classical TNF-like cytokines, necroptosis induced by proteasome inhibitors does not require caspase inhibition. However, an intact RIP homotypic interaction motif (RHIM) is essential. Surprisingly, when recruitment of MLKL to RIPK3 is restricted, proteasome inhibitors induced RIPK3-dependent apoptosis. Proteasome inhibition led to accumulation of K48-linked ubiquitinated RIPK3, which was partially reduced when Lys-264 was mutated. Taken together, these results reveal the ubiquitin-proteasome system as a novel regulatory mechanism for RIPK3-dependent necroptosis.

Topics: Animals; Antineoplastic Agents; Bortezomib; Cell Death; Cell Line; Cell Line, Tumor; Humans; Leupeptins; Mice; Neoplasms; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Kinases; Receptor-Interacting Protein Serine-Threonine Kinases; Ubiquitination

The transcriptional repressor Slug is best known to control epithelial-mesenchymal transition (EMT) and promote cancer invasion/metastasis. In this study, we demonstrate that Slug is temporally regulated during cell cycle progression. At G1/S transition, cyclin E-cyclin-dependent kinase 2 mediates the phosphorylation of Slug at Ser-54 and Ser-104, resulting in its ubiquitylation and degradation. Non-phosphorylatable Slug is markedly stabilized at G1/S transition compared with wild-type Slug and greatly leads to downregulation of DNA synthesis and checkpoint-related proteins, including TOP1, DNA Ligase IV and Rad17, reduces cell proliferation, delays S-phase progression and contributes to genome instability. Our results indicate that Slug has multifaceted roles in cancer progression by controlling both EMT and genome stability.

Topics: Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin E; Cyclin-Dependent Kinase 2; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Genomic Instability; HEK293 Cells; HeLa Cells; Humans; Leupeptins; Neoplasms; Phosphorylation; Snail Family Transcription Factors; Transcription Factors; Ubiquitination

Anti-cancer drug development typically utilizes high-throughput screening with two-dimensional (2D) cell culture. However, 2D culture induces cellular characteristics different from tumors in vivo, resulting in inefficient drug development. Here, we report an innovative high-throughput screening system using nanoimprinting 3D culture to simulate in vivo conditions, thereby facilitating efficient drug development. We demonstrated that cell line-based nanoimprinting 3D screening can more efficiently select drugs that effectively inhibit cancer growth in vivo as compared to 2D culture. Metabolic responses after treatment were assessed using positron emission tomography (PET) probes, and revealed similar characteristics between the 3D spheroids and in vivo tumors. Further, we developed an advanced method to adopt cancer cells from patient tumor tissues for high-throughput drug screening with nanoimprinting 3D culture, which we termed Cancer tissue-Originated Uniformed Spheroid Assay (COUSA). This system identified drugs that were effective in xenografts of the original patient tumors. Nanoimprinting 3D spheroids showed low permeability and formation of hypoxic regions inside, similar to in vivo tumors. Collectively, the nanoimprinting 3D culture provides easy-handling high-throughput drug screening system, which allows for efficient drug development by mimicking the tumor environment. The COUSA system could be a useful platform for drug development with patient cancer cells.

Topics: Animals; Antineoplastic Agents; Biological Assay; Cell Culture Techniques; Cell Hypoxia; Cell Line, Tumor; Drug Design; Drug Screening Assays, Antitumor; Humans; Leupeptins; Male; Mice, Inbred BALB C; Mice, Nude; Nanotechnology; Neoplasms; Spheroids, Cellular; Tumor Microenvironment

The ubiquitin-proteasome system is central in the regulation of cellular proteins controlling cell cycle progression and apoptosis, drawing much interest for developing effective targeted cancer therapies. Herein, we developed a novel pH-responsive polymeric-micelle-based carrier system to effectively deliver the proteasome inhibitor MG132 into cancer cells. MG132 is covalently bound to the block copolymer composed of polyethylene glycol (PEG) and polyaspartate through an acid-labile hydrazone bond. This bond is stable at physiological condition, but hydrolytically degradable in acidic compartments in the cell, such as late-endosomes and lysosomes, and thus, it was used for controlled release of MG132 after EPR-mediated preferential accumulation of the micelles into the tumor. MG132-loaded micelles have monodispersed size distribution with an average diameter of 45nm, and critical micelle concentration is well below 10(-7)M. In vitro studies against several cancer cell lines confirmed that MG132-loaded micelles retained the cytotoxic effect, and this activity was indeed due to the inhibition of proteasome by released MG132 from the micelles. Real-time in vitro confocal-microscopy experiments clearly indicated that MG132-conjugated micelles disintegrated only inside the target cells. By intravital confocal micro-videography, we also confirmed the prolonged circulation of MG132 loaded micelles in the bloodstream, which lead to tumor specific accumulation of micelles, as confirmed by in vivo imaging 24h after injection. These micelles showed significantly lower in vivo toxicity than free MG132, while achieving remarkable antitumor effect against a subcutaneous HeLa-luc tumor model. Our findings create a paradigm for future development of polymeric-micelle-based carrier system for other peptide aldehyde type proteasome inhibitors to make them effective cohort of the existing cancer therapeutic regiments.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Delayed-Action Preparations; Female; Humans; Hydrogen-Ion Concentration; Leupeptins; Mice, Inbred BALB C; Micelles; Neoplasms; Polymers; Proteasome Inhibitors

Rab7 plays an important role in regulating endocytic traffic. In view of an emerging role of membrane traffic in signaling and diseases, we have examined the possible role of Rab7 in oncogenesis. The role of Rab7 was investigated using shRNA-mediated knockdown in A431 and MCF7 cancer cells. To our surprise, Rab7 knockdown effectively suppressed anchorage-independent growth of cancer cells in soft agar. Anoikis (matrix-detachment triggered apoptosis) was enhanced, while the level of phosphorylated (active) Akt (which is a key survival factor) was significantly reduced. Also intriguing was the observation that EGFR and Her2 levels were significantly reduced when Rab7 was knocked-down. More robust reduction of EGFR and Her2 levels was observed when knocked-down cells were treated with HSP90 inhibitor geldanamycin (GA). Low concentration of GA (50-100 nm)-induced apoptosis of the Rab7 knocked-down cells but not control cells, suggesting that Rab7 and HSP90 together contribute to the optimal stability of EGFR and Her2 as well as to protect cancer cells from apoptosis. Rab7 seems to protect EGFR and Her2 from proteosome-mediated degradation. These results suggest that Rab7 is likely involved in protecting EGFR and Her2 from being degraded by the proteosome and in maintaining optimal Akt survival signal (especially during cell detachment or when HSP90 is inhibited). Rab7 is potentially a novel target for combinatory therapy with Hsp90 inhibitors.

Topics: Animals; Anoikis; Apoptosis; Benzoquinones; Cell Proliferation; Cell Survival; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; ErbB Receptors; Genetic Vectors; HeLa Cells; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Lentivirus; Leupeptins; Mice; Mice, Nude; Neoplasm Transplantation; Neoplasms; Phosphorylation; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Stability; Proto-Oncogene Proteins c-akt; rab GTP-Binding Proteins; rab7 GTP-Binding Proteins; Receptor, ErbB-2; RNA Interference; Signal Transduction; Time Factors; Transduction, Genetic; Tumor Burden

Propyl gallate (PG) used in processed food and medicinal preparations has been shown to induce cell death in normal and cancer cells. The inhibition of proteasome function has emerged as a useful strategy to maneuver apoptosis. Here, we investigated the combined effects of PG and MG132 (a proteasome inhibitor) on HeLa cells in relation to cell growth, cell death, reactive oxygen species (ROS) and glutathione (GSH). PG induced growth inhibition and apoptosis in HeLa cells, accompanied by the loss of mitochondrial membrane potential (MMP; ΔΨm), activation of caspase 3 and PARP cleavage. The levels of ROS and GSH depletion were increased in PG-treated HeLa cells. MG132 intensified apoptosis and PARP cleavage in PG-treated HeLa cells. MG132 also increased ROS levels including mitochondrial O2•-, MMP (ΔΨm) loss and GSH depletion in PG-treated HeLa cells. PG induced a G1 phase arrest of the cell cycle in HeLa cells, which was significantly prevented by MG132. MG132 alone inhibited HeLa cell growth via inducing the cell cycle arrests and triggering apoptosis. Conclusively, the inhibition of proteasome by MG132 plays a role as an enhancement factor in PG-induced apoptosis of HeLa cells via increasing ROS levels and GSH depletion.

Topics: Antioxidants; Apoptosis; Cell Cycle; Cysteine Proteinase Inhibitors; Drug Evaluation, Preclinical; Drug Synergism; Glutathione; HeLa Cells; Humans; Leupeptins; Membrane Potential, Mitochondrial; Neoplasms; Propyl Gallate; Proteasome Inhibitors; Reactive Oxygen Species

The accumulation of damaged or misfolded proteins, if unresolved, can lead to a detrimental consequence within cells termed proteotoxicity. Since cancerous cells often display elevated protein synthesis and by-product disposal, inhibition of the protein degradation pathways is an emerging approach for cancer therapy. However, the molecular mechanism underlying proteotoxicity remains largely unclear. We show here that inhibition of proteasomal degradation results in an increased oligomerization and activation of caspase-8 on the cytosolic side of intracellular membranes. This enhanced caspase-8 oligomerization and activation are promoted through its interaction with the ubiquitin-binding protein SQSTM1/p62 and the microtubule-associated protein light chain 3 (LC3), which are enriched at intracellular membranes in response to proteotoxic stress. Silencing LC3 by shRNA, or the LC3 mutants defective in membrane localization or p62 interaction fail to induce caspase-8 activation and apoptosis. Our results unveiled a previously unknown mechanism through which disruption of protein homeostasis induces caspase-8 oligomerization, activation, and apoptosis.

Topics: Adaptor Proteins, Signal Transducing; Animals; Apoptosis; Caspase 8; Cell Line; Cell Line, Tumor; Enzyme Activation; Fluorescent Antibody Technique; Heat-Shock Proteins; Humans; Intracellular Membranes; Leupeptins; Mice; Microtubule-Associated Proteins; Neoplasms; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Folding; Protein Multimerization; RNA Interference; RNA, Small Interfering; Sequestosome-1 Protein; Ubiquitination

Celecoxib is a COX-2 inhibitor that reduces the risk of colon cancer. However, the basis for its cancer chemopreventive activity is not fully understood. In this study, we defined a mechanism of celecoxib action based on degradation of cellular FLICE-inhibitory protein (c-FLIP), a major regulator of the death receptor pathway of apoptosis. c-FLIP protein levels are regulated by ubiquitination and proteasome-mediated degradation. We found that celecoxib controlled c-FLIP ubiquitination through Akt-independent inhibition of glycogen synthase kinase-3 (GSK3), itself a candidate therapeutic target of interest in colon cancer. Celecoxib increased the levels of phosphorylated GSK3, including the α and β forms, even in cell lines, where phosphorylated Akt levels were not increased. Phosphoinositide 3-kinase inhibitors abrogated Akt phosphorylation as expected but had no effect on celecoxib-induced GSK3 phosphorylation. In contrast, protein kinase C (PKC) inhibitors abolished celecoxib-induced GSK3 phosphorylation, implying that celecoxib influenced GSK3 phosphorylation through a mechanism that relied upon PKC and not Akt. GSK3 blockade either by siRNA or kinase inhibitors was sufficient to attenuate c-FLIP levels. Combining celecoxib with GSK3 inhibition enhanced attenuation of c-FLIP and increased apoptosis. Proteasome inhibitor MG132 reversed the effects of GSK3 inhibition and increased c-FLIP ubiquitination, confirming that c-FLIP attenuation was mediated by proteasomal turnover as expected. Our findings reveal a novel mechanism through which the regulatory effects of c-FLIP on death receptor signaling are controlled by GSK3, which celecoxib acts at an upstream level to control independently of Akt.

Topics: Apoptosis; CASP8 and FADD-Like Apoptosis Regulating Protein; Celecoxib; Cell Line, Tumor; Cyclooxygenase 2 Inhibitors; Glycogen Synthase Kinase 3; Humans; Leupeptins; Neoplasms; Phosphorylation; Proteasome Endopeptidase Complex; Protein Kinase C; Proteolysis; Proto-Oncogene Proteins c-akt; Pyrazoles; Receptors, Death Domain; RNA, Small Interfering; Sulfonamides; Ubiquitination

Proteasome inhibition is a promising approach for cancer therapy. However, the mechanisms involved have not been fully elucidated. Gap junctions play important roles in the regulation of tumor cell phenotypes and mediation of the bystander effect in cancer therapy. Because the degradation of gap junction proteins involves the proteasome, we speculated that altered gap junctions might contribute to the antitumor activities of proteasome inhibition. Incubation of Hepa-1c1c7 cells with the proteasome inhibitor MG132 elevated the levels of gap junction protein connexin 43 (Cx43) and promoted gap junctional intercellular communication. This was associated with a marked accumulation of ubiquitylated Cx43 and a significantly decreased rate of Cx43 degradation. The elevated Cx43 contributed to MG132-induced cell apoptosis. This is shown by the observations that: (i) overexpression of Cx43 in the gap junction-deficient LLC-PK1 cells rendered them vulnerable to MG132-elicited cell injury; (ii) fibroblasts derived from Cx43-null mice were more resistant to MG-132 compared with Cx43 wild-type control; and (iii) the gap junction inhibitor flufenamic acid significantly attenuated cell damage caused by MG132 in Hepa-1c1c7 cells. Further studies demonstrated that MG132 activates endoplasmic reticulum stress. Exposure of cells to the endoplasmic reticulum stress inducers thapsigargin and tunicamycin also led to cell apoptosis, which was modulated by Cx43 levels in a way similar to MG132. These results suggested that elevated Cx43 sensitizes cells to MG132-induced cell apoptosis. Regulation of gap junctions could be an important mechanism behind the antitumor activities of proteasome inhibitors.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Connexin 43; Cysteine Proteinase Inhibitors; Endoplasmic Reticulum; Gap Junctions; Leupeptins; LLC-PK1 Cells; Mice; Neoplasms; Swine

ACK1 (activated Cdc42-associated kinase 1) is a cytoplasmic tyrosine kinase implicated in trafficking through binding to epidermal growth factor (EGF) receptor and clathrin. Here, we have identified a new ACK1-binding partner, the E3 ubiquitin ligase Nedd4-2, which binds ACK1 via a conserved PPXY-containing region. We show that this motif also binds Nedd4-related proteins and several other WW domain-containing proteins, including the tumor suppressor oxidoreductase Wwox. In HeLa cells ACK1 colocalizes with Nedd4-2 in clathrin-rich vesicles, requiring this PPXY motif. Nedd4-2 strongly down-regulates ACK1 levels when coexpressed, and this process can be blocked by proteasome inhibitor MG132. ACK1 degradation via Nedd4 requires their mutual interaction and a functional E3 ligase; it is also driven by ACK1 activity. ACK1 is polyubiquitinated in vivo, and dominant inhibitory Nedd4 blocks endogenous ACK1 turnover in response to acute EGF treatment. Because EGF stimulation activates ACK1 ( Galisteo, M., Y., Y., Urena, J., and Schlessinger, J. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 9796-9801 ), our result suggest that EGF receptor-mediated ACK1 activation allows Nedd4-2 to drive kinase degradation. Thus the interplay between Nedd4-2-related E3 ligases that regulate ACK1 levels and Cbl that modifies EGF receptor impinges on cell receptor dynamics. These processes are particularly pertinent given the report of genomic amplification of the ACK1 locus in metastatic tumors.

Topics: Amino Acid Motifs; Animals; Chlorocebus aethiops; Clathrin; Clathrin-Coated Vesicles; COS Cells; Cysteine Proteinase Inhibitors; Down-Regulation; Endosomal Sorting Complexes Required for Transport; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Enzymologic; HeLa Cells; Humans; Leupeptins; Nedd4 Ubiquitin Protein Ligases; Neoplasm Metastasis; Neoplasms; Oxidoreductases; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Binding; Protein Structure, Tertiary; Protein Transport; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-cbl; Tumor Suppressor Proteins; Ubiquitin-Protein Ligases; Ubiquitination; WW Domain-Containing Oxidoreductase

This paper reports the synthesis of compounds formed by two indole systems separated by a heterocycle (pyridine or piperazine). As a primary screening, the new compounds were submitted to the National Cancer Institute for evaluation of antitumor activity in the human cell line screen. The pyridine derivatives were far more active than the piperazine derivatives. For the study of the mechanism of action, the most active compounds were subjected to COMPARE analysis and to further biological tests including proteasome inhibition and inhibition of plasma membrane electron transport. The compound bearing the 5-methoxy-2-indolinone moiety was subjected to the first in vivo experiment (hollow fiber assay) and was active. It was therefore selected for the second in vivo experiment (human tumor xenograft in mice). In conclusion we demonstrated that this approach was successful, since some of the compounds described are much more active than the numerous, so far prepared and tested 3-indolylmethylene-2-indolinones.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Indoles; Molecular Structure; Neoplasms; Ubiquitin-Protein Ligases

Belactosin A is a potent proteasome inhibitor isolated from Streptomyces metabolites. Here we show that a hydrophobic belactosin A derivative, dansyl-KF33955, can covalently, and specifically, affinity label the catalytic subunits of the 26S proteasome, which consists of the 20S protein degrading core particle and the 19S regulatory particles. The labeling of catalytic subunits proceeds faster in intact proteasomes in vivo than in isolated 20S core particles. These data suggest that the 19S regulatory particle may facilitate entry of the inhibitor into the 20S core particle. This cell-permeable chemical probe is an excellent tool with which to study the interactions of this proteasome inhibitor with proteasomes in intact cells.

Topics: Antineoplastic Agents; Catalytic Domain; Dipeptides; Drug Design; Erythrocytes; HeLa Cells; Humans; Inhibitory Concentration 50; Intercellular Signaling Peptides and Proteins; Lactones; Models, Chemical; Neoplasms; Peptides; Proteasome Endopeptidase Complex; Tandem Mass Spectrometry

Ecteinascidin 743 (Et743; trabectedin, Yondelis) has recently been approved in Europe for the treatment of soft tissue sarcomas and is undergoing clinical trials for other solid tumors. Et743 selectively targets cells proficient for TC-NER, which sets it apart from other DNA alkylating agents. In the present study, we examined the effects of Et743 on RNA Pol II.. We report that Et743 induces the rapid and massive degradation of transcribing Pol II in various cancer cell lines and normal fibroblasts. Pol II degradation was abrogated by the proteasome inhibitor MG132 and was dependent on TC-NER. Cockayne syndrome (CS) cells and xeroderma pigmentosum (XP) cells (XPD, XPA, XPG, and XPF) were defective in Pol II degradation, whereas XPC cells whose defect is limited to global genome NER in nontranscribing regions were proficient for Pol II degradation. Complementation of the CSB and XPD cells restored Pol II degradation. We also show that cells defective for the VHL complex were defective in Pol II degradation and that complementation of those cells restores Pol II degradation. Moreover, VHL deficiency rendered cells resistant to Et743-induced cell death, a similar effect to that of TC-NER deficiency.. These results suggest that both TC-NER-induced and VHL-mediated Pol II degradation play a role in cell killing by Et743.

Topics: Antineoplastic Agents, Alkylating; Blotting, Western; Cell Survival; Cells, Cultured; Cockayne Syndrome; Cysteine Proteinase Inhibitors; Dioxoles; DNA Damage; DNA Helicases; DNA Repair; DNA Repair Enzymes; DNA-Binding Proteins; Fibroblasts; Genetic Complementation Test; Humans; Leupeptins; Neoplasms; Phosphorylation; Poly-ADP-Ribose Binding Proteins; RNA Polymerase II; Sarcoma; Tetrahydroisoquinolines; Trabectedin; Transcription, Genetic; von Hippel-Lindau Disease; Von Hippel-Lindau Tumor Suppressor Protein; Xeroderma Pigmentosum; Xeroderma Pigmentosum Group D Protein

Hypoxia-inducible factor 1 (HIF-1) plays an essential role in tumor angiogenesis and growth by regulating the transcription of several genes in response to hypoxic stress and changes in growth factors. This study was designed to investigate the effects of deguelin on tumor growth and angiogenesis, and the mechanisms underlying the antitumor activities of deguelin. We show here that orally administered deguelin inhibits tumor growth and blocks tumor angiogenesis in mice. Deguelin decreased expression of HIF-1alpha protein and its target genes, such as VEGF, in a subset of cancer cell lines, including H1299 lung cancer cells, and vascular endothelial cells in normoxic and hypoxic conditions. Overexpression of vascular endothelial growth factor by adenoviral vector infection abolished the antiangiogenic effects of deguelin on H1299 nonsmall cell lung cancer cells. Deguelin inhibited de novo synthesis of HIF-1alpha protein and reduced the half-life of the synthesized protein. MG132, a proteasome inhibitor, protected the hypoxia- or IGF-induced HIF-1alpha protein from deguelin-mediated degradation. Our findings suggest that deguelin is a promising antiangiogenic therapeutic agent in cancer targeting HIF-1alpha. Considering that HIF-1alpha is overexpressed in a majority of human cancers, deguelin could offer a potent therapeutic agent for cancer.

Topics: Angiogenesis Inhibitors; Animals; Animals, Genetically Modified; Aorta; Blotting, Western; Cell Hypoxia; Cell Movement; Cell Proliferation; Chick Embryo; Chorioallantoic Membrane; Collagen; Culture Media, Conditioned; Cysteine Proteinase Inhibitors; Drug Combinations; Endothelium, Vascular; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Laminin; Leupeptins; Luciferases; Mice; Neoplasms; Neovascularization, Pathologic; Proteasome Endopeptidase Complex; Proteoglycans; Reverse Transcriptase Polymerase Chain Reaction; Rotenone; Tumor Cells, Cultured; Ubiquitin; Vascular Endothelial Growth Factor A; Zebrafish

When a replicative DNA polymerase encounters a lesion on the template strand and stalls, it is replaced with another polymerase(s) with low processivity that bypasses the lesion to continue DNA synthesis. This phenomenon is known as translesion replication or replicative bypass. Failing this, the cell is increasingly likely to undergo apoptosis. In this study, we found that proteasome inhibitors prevent translesion replication in human cancer cells but not in normal cells. Three proteasome inhibitors, MG-132, lactacystin, and MG-262, inhibited UV-induced translesion replication in a wide range of cancer cell lines, including HeLa, HGC-27, MCF-7, HepG2, WiDr, a malignant melanoma, an acute lymphoblastic leukemia, and a multiple myeloma cell line; irrespective of cell origin, histological type, or p53 status. In contrast, these inhibitors had little or no influence on normal fibroblasts (NB1RGB and TIG-1) or a normal liver mesenchymal (LI90) cell line. Among the DNA-damaging antineoplastic agents, cisplatin caused a UV-type translesion reaction; the proteasome inhibitors delayed cisplatin-induced translesion replication in cancer cell lines but had only a weak effect on normal cell lines. Therefore, translesion replication would be an effective target of proteasome inhibitors for cancer chemotherapy by which cancer cells can be efficiently sensitized to DNA-damaging antineoplastic agents, such as cisplatin.

Topics: Acetylcysteine; Boronic Acids; Caffeine; Cells, Cultured; Cisplatin; Cysteine Proteinase Inhibitors; DNA Damage; DNA Repair; DNA Replication; HeLa Cells; Humans; Leupeptins; Neoplasms; Proteasome Inhibitors; Tumor Cells, Cultured; Ultraviolet Rays

c-IAP1, a member of the inhibitor of apoptosis protein (IAP) family and a RING finger ubiquitin ligase (E3), has been proposed to be an important oncogene. In many types of cancers, the levels of c-IAP1 are upregulated, which contributes positively to tumorigenesis. However, the mechanism by which c-IAP1 promotes tumorigenesis has proven elusive. Although proteins in the IAP family may function as caspase inhibitors, c-IAP1 was shown to be a poor inhibitor of caspases. Here we show that c-IAP1 catalyzes ubiquitination of Max-dimerization protein-1 (Mad1), a cellular antagonist of Myc. Ubiquitination of Mad1 by c-IAP1 accelerates its degradation by the 26S proteasome pathway, and this reduction of the Mad1 levels cooperates with Myc to promote cell proliferation. Our results demonstrate that c-IAP1 exerts its oncogenic functions by promoting the degradation of an important negative regulator in the Myc pathway.

Topics: Apoptosis; Cell Cycle Proteins; Cell Proliferation; Cysteine Proteinase Inhibitors; Humans; Inhibitor of Apoptosis Proteins; Leupeptins; Neoplasms; Nuclear Proteins; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Proto-Oncogene Proteins c-myc; Signal Transduction; Tumor Stem Cell Assay; Ubiquitin; Ubiquitin-Protein Ligases; Ubiquitination

Smad4 is a key signal transducer of the transforming growth factor-beta (TGF-beta) superfamily of growth factors that are critical regulators of embryonic patterning and adult tissue homeostasis. The biological activity of the TGF-beta signaling is tightly controlled at multiple levels, including the abundance of SMAD4 proteins. We previously recovered a novel allele of Smad4 in a gene-based screen in N-ethyl-N-nitrosourea (ENU)-mutagenized mouse embryonic stem cells. The mutation resulted in an unstable truncated protein that is degraded through proteasomal pathways. In the heterozygous state, this allele acts in a dominant negative fashion to reduce the wild-type protein level as well as signaling output. Biochemical characterization indicated that the truncated protein is able to form a complex with the wild-type protein, thus targeting it for proteasomal degradation as well. Phenotypic analyses of the heterozygous animals provided insight into the threshold requirement of Smad4-dependent signaling in vivo.

Topics: Alleles; Animals; Cycloheximide; Cysteine Proteinase Inhibitors; Embryo, Mammalian; Genes, Dominant; Heterozygote; Leupeptins; Mice; Mice, Knockout; Mutation; Neoplasms; Phenotype; Protein Synthesis Inhibitors; Signal Transduction; Smad4 Protein; Stem Cells

Non-small cell lung cancer frequently presents as a locally advanced disease. In this setting, radiation has a prominent role in cancer therapy. However, tumor adaptation to oxidative stress may lessen the efficacy of radiation therapy. Recent studies demonstrate that proteasome inhibitors increase the efficacy of radiation against a range of tumors. Although proteasome inhibition impacts on NF-kappaB translocation, the precise mechanism through which proteasome inhibitors induce tumor cell death and promote radiation efficacy remains unclear. The purpose of this study is to evaluate the potential of the proteasome inhibitor, MG-132, to improve the efficacy of radiation therapy and to determine whether its effect is linked to the suppression of the antioxidant enzyme, manganese superoxide dismutase (MnSOD). Human NSCLC (A549) cells were utilized both in vivo and in vitro to evaluate proteasome inhibition on radiation response. In vivo, mice that received combined treatments of 2.5 microg/g body weight MG-132 and 30 Gy demonstrated a delay in tumor regrowth in comparison to the 30 Gy control group. In vitro, clonegenic survival assays confirmed a dose-dependent enhancement of radiation sensitivity in combination with MG-132 and a significant interaction between the two. The levels of IkappaB-alpha, a NF-kappaB target gene and also an inhibitor of NF-kappaB nuclear translocation, decreased in a time-dependent manner following administration of MG-132 confirming the inhibition of the 26S proteasome. The MnSOD protein level was increased consistent with lower levels of IkappaB-alpha, confirming a NF-kappaB-mediated effect. Cells treated with radiation demonstrated an induction of MnSOD; however, the administration of MG-132 suppressed this induction These results support the hypothesis that proteasome inhibitors such as MG-132 can increase the efficacy of radiation therapy, in part, by suppression of cytoprotective NF-kappaB-mediated MnSOD expression.

Topics: Active Transport, Cell Nucleus; Animals; Antioxidants; Blotting, Western; Body Weight; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Combined Modality Therapy; Dose Fractionation, Radiation; Dose-Response Relationship, Drug; Female; Humans; I-kappa B Proteins; Leupeptins; Lung Neoplasms; Mice; Mice, Nude; Neoplasm Transplantation; Neoplasms; NF-kappa B; NF-KappaB Inhibitor alpha; Oxidative Stress; Protease Inhibitors; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Transport; Radiation Tolerance; Reactive Oxygen Species; Superoxide Dismutase; Time Factors

The heat shock proteins (HSPs) are molecular chaperones that are emerging as biochemical regulators of cell growth, apoptosis, protein homeostasis and intracellular targeting of peptides. The immunological function of the HSPs are imparted by tissue specific peptides associated with the HSPs and as such autologous cancer derived HSP-peptide complexes are unique therapeutic agents. Since a majority of the intracellular peptides are generated by the proteasome, we examined the consequence of abrogation of proteasome function by proteasome inhibitors (PIs) such as Lactacystin, MG-132 and LLM on the growth and induction profile of HSP70 and gp96 using hematopoietic, lymphoid, and epithelial derived cancer cell lines. The effect on growth was measured by the XTT assay and induction of the heat shock proteins by western blot analyses using HSP70 and gp96 specific antibodies. Of the PIs tested, cancer cells, were most sensitive to MG-132 and least sensitive to LLM. MG-132 also showed a 10-fold differential sensitivity between estrogen receptor positive, (ER+) MCF-7 cells and negative cells, (ER-) MDA-MB-231. Induction of heat shock proteins, gp96 and HSP70 was, however, noted in response to LLM. Since LLM exhibited minimal cytotoxic effect, metabolic stress that results in induction of HSPs may not be translated in cell growth inhibition and that there may exist a cell-type specific phenomenon in the HSP response to PI mediated metabolic stress.

Topics: Acetylcysteine; Antigens, Neoplasm; Blotting, Western; Cell Division; Cell Line; Cell Survival; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Heat-Shock Proteins; HL-60 Cells; Humans; K562 Cells; Leupeptins; Multienzyme Complexes; Neoplasms; Proteasome Endopeptidase Complex; Tumor Cells, Cultured

The human multidrug resistance P-glycoprotein (P-gp) contributes to the phenomenon of multidrug resistance during cancer and AIDS chemotherapy. A potential novel strategy to circumvent the effects of P-gp during chemotherapy is to prevent maturation of P-gp during biosynthesis so that the transporter does not reach the cell surface. Here we report that immature, core-glycosylated P-gp that is prevented from reaching the cell surface by processing mutations or by proteasome inhibitors such as lactacystin or MG-132 exhibited no detectable drug-stimulated ATPase activity. Disulfide cross-linking analysis also showed that the immature P-gp did not exhibit ATP-induced conformational changes as found in the mature enzyme. In addition, the immature P-gp was more sensitive to trypsin than the mature enzyme. These results suggest that P-gp is unlikely to be functional immediately after synthesis. These differences in the structural and enzymatic properties of the mature and core-glycosylated, immature P-gp could potentially be used during chemotherapy, and should result in the search for compounds that can specifically inhibit the maturation of P-gp.

Topics: Acetylcysteine; Acquired Immunodeficiency Syndrome; Adenosine Triphosphatases; Adenosine Triphosphate; ATP Binding Cassette Transporter, Subfamily B, Member 1; Biological Transport; Cell Membrane; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Drug Resistance, Multiple; Glycosylation; Humans; Leupeptins; Multienzyme Complexes; Neoplasms; Proteasome Endopeptidase Complex; Protein Conformation; Protein Folding; Protein Precursors; Protein Processing, Post-Translational

Many cells are resistant to stimuli that can induce apoptosis, but the mechanisms involved are not fully understood. The activation of the transcription factor nuclear factor-kappa B (NF-kappaB) by tumor necrosis factor (TNF), ionizing radiation, or daunorubicin (a cancer chemotherapeutic compound), was found to protect from cell killing. Inhibition of NF-kappaB nuclear translocation enhanced apoptotic killing by these reagents but not by apoptotic stimuli that do not activate NF-kappaB. These results provide a mechanism of cellular resistance to killing by some apoptotic reagents, offer insight into a new role for NF-kappaB, and have potential for improvement of the efficacy of cancer therapies.

Topics: Antibiotics, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Nucleus; Cycloheximide; Daunorubicin; DNA-Binding Proteins; Humans; I-kappa B Proteins; Interleukin-1; Leupeptins; Neoplasms; NF-kappa B; NF-KappaB Inhibitor alpha; Protein Synthesis Inhibitors; Radiation, Ionizing; Staurosporine; Transcription Factor RelA; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha