ABSTRACT
CIGB-552 is a synthetic anti-tumor peptide capable of reducing tumor size and increasing the lifespan of tumor-bearing mice. Part of its anti-cancer effects consists of inducing apoptosis, modulating NF-kB signaling pathway, and the angiogenesis process. Although one of its major mediators, the COMMD1 protein, has been identified, the mechanism by which CIGB-552 exerts such effects remains elusive. In the present study, we show the role of COMMD1 in CIGB-552 mechanism of action by generating the COMMD1 knock-out from the human lung cancer cell line NCI-H460. A microarray was performed to analyze both wild-type and KO cell lines with regard to CIGB-552 treatment. Additionally, different signaling pathways were studied in both cell lines to validate the results. Furthermore, the interaction between CIGB-552 and COMMD1 was analyzed by confocal microscopy. By signaling pathway analysis we found that genes involved in cell proliferation and apoptosis, oncogenic transformation, angiogenesis and inflammatory response are potentially regulated by the treatment with CIGB-552. We then demonstrated that CIGB-552 is capable of modulating NF-kB in both 2D and 3D cell culture models. Finally, we show that the ability of CIGB-552 to negatively modulate NF-kB and HIF-1 pathways is impaired in the COMMD1 knock-out NCI-H460 cell line, confirming that COMMD1 is essential for the peptide mechanism of action.
Subject(s)
Adaptor Proteins, Signal Transducing/physiology , Angiogenesis Inhibitors/pharmacology , Anti-Inflammatory Agents/pharmacology , Cell-Penetrating Peptides/pharmacology , Colonic Neoplasms/drug therapy , Inflammation/drug therapy , Lung Neoplasms/drug therapy , Neovascularization, Pathologic/drug therapy , Animals , Apoptosis , Cell Proliferation , Colonic Neoplasms/blood supply , Colonic Neoplasms/immunology , Colonic Neoplasms/metabolism , Gene Expression Regulation, Neoplastic , Humans , Inflammation/immunology , Inflammation/metabolism , Inflammation/pathology , Lung Neoplasms/blood supply , Lung Neoplasms/immunology , Lung Neoplasms/metabolism , Mice , Mice, Knockout , NF-kappa B/metabolism , Neovascularization, Pathologic/immunology , Neovascularization, Pathologic/metabolism , Neovascularization, Pathologic/pathology , Tumor Cells, CulturedABSTRACT
CIGB-552 is a twenty-amino-acid novel synthetic peptide that has proven to be effective in reducing tumor size and increasing lifespan in tumor-bearing mice. Such capability is conferred by its cell-penetrating peptide character, which allows it to enter cells and elicit a pro-apoptotic effect through its major mediator, COMMD1 protein. Cell-penetrating peptides are able to use different internalization mechanisms, such as endocytosis or direct transduction through the plasma membrane. Although CIGB-552 cytotoxicity has been evaluated in several non-tumor- and tumor-derived cell lines, no data regarding the relationship between cell line sensitivity, cell penetrating capacity, the internalization mechanisms involved, COMMD1 expression levels, or its subcellular localization has yet been produced. Here, we present the results obtained from a comparative analysis of CIGB-552 sensitivity, internalization capacity and the mechanisms involved in three human tumor-derived cell lines from different origins: mammary gland, colon and lung (MCF-7, HT-29 and H460, respectively). Furthermore, cell surface markers relevant for internalization processes such as phosphatidylserine, as well as CIGB-552 target COMMD1 expression/localization, were also evaluated. We found that both endocytosis and transduction are involved in CIGB-552 internalization in the three cell lines evaluated. However, CIGB-552 incorporation efficiency and contribution of each mechanism is cell-line dependent. Finally, sensitivity was directly correlated with high internalization capacity in those cell lines where endocytosis had a major contribution on CIGB-552 internalization.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Antineoplastic Agents , Cell-Penetrating Peptides , Endocytosis/drug effects , Neoplasm Proteins/metabolism , Neoplasms/drug therapy , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/pharmacology , Cell-Penetrating Peptides/chemical synthesis , Cell-Penetrating Peptides/chemistry , Cell-Penetrating Peptides/pharmacokinetics , Cell-Penetrating Peptides/pharmacology , Humans , MCF-7 Cells , Neoplasms/metabolism , Neoplasms/pathologyABSTRACT
Using LAURDAN spectral imaging and spectral phasor analysis we concurrently studied the growth and hydration state of subcellular organelles (lamellar body-like, LB-like) from live A549 lung cancer cells at different post-confluence days. Our results reveal a time dependent two-step process governing the size and hydration of these intracellular LB-like structures. Specifically, a first step (days 1 to 7) is characterized by an increase in their size, followed by a second one (days 7 to 14) where the organelles display a decrease in their global hydration properties. Interestingly, our results also show that their hydration properties significantly differ from those observed in well-characterized artificial lamellar model membranes, challenging the notion that a pure lamellar membrane organization is present in these organelles at intracellular conditions. Finally, these LB-like structures show a significant increase in their hydration state upon secretion, suggesting a relevant role of entropy during this process.
Subject(s)
2-Naphthylamine/analogs & derivatives , Fluorescent Dyes/chemistry , Intracellular Membranes/metabolism , Laurates/chemistry , Organelles/metabolism , Water/metabolism , 2-Naphthylamine/chemistry , A549 Cells , Biological Transport , Entropy , Humans , Intracellular Membranes/ultrastructure , Organelle Size , Organelles/ultrastructure , Osmolar Concentration , Spectrometry, FluorescenceABSTRACT
Because of resistance development by cancer cells against current anticancer drugs, there is a considerable interest in developing novel antitumor agents. We have previously demonstrated that CIGB-552, a novel cell-penetrating synthetic peptide, was effective in reducing tumor size and increasing lifespan in tumor-bearing mice. Studies of protein-peptide interactions have shown that COMMD1 protein is a major mediator of CIGB-552 antitumor activity. Furthermore, a typical serine-protease degradation pattern for CIGB-552 in BALB/c mice serum was identified, yielding peptides which differ from CIGB-552 in size and physical properties. In the present study, we show the results obtained from a comparative analysis between CIGB-552 and its main metabolites regarding physicochemical properties, cellular internalization, and their capability to elicit apoptosis in MCF-7 cells. None of the analyzed metabolites proved to be as effective as CIGB-552 in promoting apoptosis in MCF-7. Taking into account these results, it seemed important to examine their cell-penetrating capacity and interaction with COMMD1. We show that internalization, a lipid binding-dependent process, is impaired as well as metabolite-COMMD1 interaction, key component of the apoptotic mechanism. Altogether, our results suggest that features conferred by the amino acid sequence are decisive for CIGB-552 biological activity, turning it into the minimal functional unit. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.
Subject(s)
Adaptor Proteins, Signal Transducing/antagonists & inhibitors , Amino Acids/chemistry , Antineoplastic Agents/pharmacology , Cell-Penetrating Peptides/pharmacology , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Amino Acid Sequence , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Apoptosis/drug effects , Biotransformation , Caspase 7/genetics , Caspase 7/metabolism , Cell Proliferation/drug effects , Cell-Penetrating Peptides/chemical synthesis , Cell-Penetrating Peptides/chemistry , Gene Expression , Humans , MCF-7 Cells , Mice , Mice, Inbred BALB C , Molecular Dynamics Simulation , Protein Binding , Structure-Activity RelationshipABSTRACT
The NF-κB is a transcription factor which plays a key role in regulating biological processes. In response to signals, NF-κB activation occurs via phosphorylation of its inhibitor, which dissociates from the NF-κB dimer allowing the translocation to the nucleus, inducing gene expression. NF-κB activation has direct screening applications for drug discovery for several therapeutic indications. Thus, pathway-specific reporter cell systems appear as useful tools to screen and unravel the mode of action of probiotics and natural and synthetic compounds. Here, we describe the generation, characterization, and validation of human epithelial reporter cell lines for functional studies of NF-κB activation by different pro- and anti-inflammatory agents. Caco-2 and HT-29 cells were transfected with a pNF-κB-hrGFP plasmid which contains the GFP gene under the control of NF-κB binding elements. Three proinflammatory cytokines (TNF-α, IL-1ß, and LPS) were able to activate the reporter systems in a dose-response manner, which corresponds to the activation of the NF-κB signaling pathway. Finally, the reporter cell lines were validated using lactic acid bacteria and a natural compound. We have established robust Caco-2-NF-κB-hrGFP and HT-29-NF-κB-hrGFP reporter cell lines which represent a valuable tool for primary screening and identification of bacterial strains and compounds with a potential therapeutic interest.
Subject(s)
NF-kappa B/physiology , Caco-2 Cells , Cell Polarity , Fluorescent Antibody Technique , HT29 Cells , Humans , Toll-Like Receptor 4/analysis , Transcription Factor RelA/analysisABSTRACT
Primary cilia are conserved organelles that play crucial roles as mechano- and chemosensors, as well as transducing signaling cascades. Consequently, ciliary dysfunction results in a broad range of phenotypes: the ciliopathies. Bardet-Biedl syndrome (BBS), a model ciliopathy, is caused by mutations in 16 known genes. However, the biochemical functions of the BBS proteins are not fully understood. Here we show that the BBS7 protein (localized in the centrosomes, basal bodies and cilia) probably has a nuclear role by virtue of the presence of a biologically confirmed nuclear export signal. Consistent with this observation, we show that BBS7 interacts physically with the polycomb group (PcG) member RNF2 and regulate its protein levels, probably through a proteasome-mediated mechanism. In addition, our data supports a similar role for other BBS proteins. Importantly, the interaction with this PcG member is biologically relevant because loss of BBS proteins leads to the aberrant expression of endogenous RNF2 targets in vivo, including several genes that are crucial for development and for cellular and tissue homeostasis. Our data indicate a hitherto unappreciated, direct role for the BBS proteins in transcriptional regulation and potentially expand the mechanistic spectrum that underpins the development of ciliary phenotypes in patients.
Subject(s)
Gene Expression Regulation , Proteins/physiology , Transcription, Genetic , Adaptor Proteins, Signal Transducing , Animals , Cell Nucleus/metabolism , Computer Simulation , Cytoskeletal Proteins , HEK293 Cells , HeLa Cells , Humans , Mice , NIH 3T3 Cells , Nuclear Export Signals , Polycomb Repressive Complex 1/metabolism , Protein Transport , Proteins/metabolism , Zebrafish/geneticsABSTRACT
Accumulation of the COMMD1 protein as a druggable pharmacology event to target cancer cells has not been evaluated so far in cancer animal models. We have previously demonstrated that a second-generation peptide, with cell-penetrating capacity, termed CIGB-552, was able to induce apoptosis mediated by stabilization of COMMD1. Here, we explore the antitumor effect by subcutaneous administration of CIGB-552 in a therapeutic schedule. Outstandingly, a significant delay of tumor growth was observed at 0.2 and 0.7 mg/kg (p < 0.01) or 1.4 mg/kg (p < 0.001) after CIGB-552 administration in both syngeneic murine tumors and patient-derived xenograft models. Furthermore, we evidenced that (131)I-CIGB-552 peptide was actually accumulated in the tumors after administration by subcutaneous route. A typical serine-proteases degradation pattern for CIGB-552 in BALB/c mice serum was identified. Further, biological characterization of the main metabolites of the peptide CIGB-552 suggests that the cell-penetrating capacity plays an important role in the cytotoxic activity. This report is the first in describing the antitumor effect induced by systemic administration of a peptide that targets COMMD1 for stabilization. Moreover, our data reinforce the perspectives of CIGB-552 for cancer targeted therapy.
Subject(s)
Antineoplastic Agents/pharmacology , Antineoplastic Agents/pharmacokinetics , Cell-Penetrating Peptides/pharmacology , Cell-Penetrating Peptides/pharmacokinetics , Neoplasms, Experimental/drug therapy , Neoplasms, Experimental/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Amino Acid Sequence , Animals , Antimicrobial Cationic Peptides/chemistry , Antineoplastic Agents/chemistry , Apoptosis/drug effects , Arthropod Proteins/chemistry , Cell Line, Tumor , Cell Survival/drug effects , Cell-Penetrating Peptides/chemistry , Female , HT29 Cells , Humans , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Neoplasms, Experimental/pathology , Protein Stability/drug effects , Tissue Distribution , Xenograft Model Antitumor AssaysABSTRACT
Biological membranes allow the regulation of numerous cellular processes, which are affected when unfavorable environmental factors are perceived. Lipids and proteins are the principal components of biological membranes. Each lipid has unique biophysical properties, and, therefore the lipid composition of the membrane is critical to maintaining the bilayer structure and functionality. Membrane composition and integrity are becoming the focus of studies aiming to understand how plants adapt to its environment. In this study, using a combination of di-4-ANEPPDHQ fluorescence and spectral phasor analysis, we report that the drought hypersensitive/squalene epoxidase (dry2/sqe1-5) mutant with reduced major sterols such as sitosterol and stigmasterol in roots presented higher membrane fluidity than the wild type. Moreover, analysis of endomembrane dynamics showed that vesicle formation was affected in dry2/sqe1-5. Further analysis of proteins associated with sterol rich micro domains showed that dry2/sqe1-5 presented micro domains function altered.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Cell Membrane/metabolism , Membrane Fluidity , Plant Roots/metabolism , Squalene Monooxygenase/metabolism , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Cell Membrane/genetics , Dehydration/metabolism , Plant Roots/genetics , Sitosterols/metabolism , Squalene Monooxygenase/genetics , Stigmasterol/metabolismABSTRACT
Until recently, synonymous mutations (which do not change amino acids) have been much neglected. Some evidence suggests that this kind of mutations could affect mRNA secondary structure or stability, translation kinetics and protein structure. To explore deeper the role of synonymous mutations, we studied their consequence on the functional activity of the estrogen receptor alpha (ERα). The ERα is a ligand-inducible transcription factor that orchestrates pleiotropic cellular effects, at both genomic and non-genomic levels in response to estrogens. In this work we analyzed in transient transfection experiments, the activity of ERα carrying the synonymous mutation Ala87, a polymorphism involving about 5-10% of the population. In comparison to the wild type receptor, our results show that ERαA87 mutation reduces the transactivation efficiency of ERα on an ERE reporter gene while its expression level remains similar. This mutation enhances 4-OHT-induced transactivation of ERα on an AP1 reporter gene. Finally, the mutation affects the subcellular localization of ERα in a cell type specific manner. It enhances the cytoplasmic location of ERα without significant changes in non-genomic effects of E2. The functional alteration of the ERαA87 determined in this work highlights the relevance of synonymous mutations for biomedical and pharmacological points of view.
Subject(s)
Estrogen Receptor alpha/metabolism , Gene Expression Regulation , Mutation/genetics , Response Elements/genetics , Transcription, Genetic , Blotting, Western , Estrogen Receptor alpha/genetics , Estrogens/pharmacology , Fluorescent Antibody Technique , HeLa Cells , Hep G2 Cells , Humans , Protein Binding , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Transcription Factor AP-1/genetics , Transcription Factor AP-1/metabolism , Transcriptional Activation/drug effectsABSTRACT
We have demonstrated that the peptide L-2 designed from an alanine scanning of the Limulus-derived LALF32-51 region is a potential candidate for the anticancer therapy and its cell-penetrating capacity is an associated useful property. By the modification in the primary structure of L-2, a second-generation peptide (CIGB-552) was developed. However, the molecular mechanism underlying its cytotoxic activity remains partially unknown. In this study, it was shown that CIGB-552 increases the levels of COMMD1, a protein involved in copper homeostasis, sodium transport, and the NF-κB signaling pathway. We found that CIGB-552 induces ubiquitination of RelA and inhibits the antiapoptotic activity regulated by NF-κB, whereas the knockdown of COMMD1 blocks this effect. We also found that CIGB-552 decreases the antioxidant capacity and induces the peroxidation of proteins and lipids in the tumor cells. Altogether, this study provides new insights into the mechanism of action of the peptide CIGB-552, which could be relevant in the design of future anticancer therapies.
ABSTRACT
There is substantial evidence suggesting that certain parasites can have antitumor properties. We evaluated mucin peptides derived from the helminth Echinococcus granulosus (denominated Egmuc) as potential inducers of antitumor activity. We present data showing that Egmuc peptides were capable of inducing an increase of activated NK cells in the spleen of immunized mice, a fact that was correlated with the capacity of splenocytes to mediate killing of tumor cells. We demonstrated that Egmuc peptides enhance LPS-induced maturation of dendritic cells in vitro by increasing the production of IL-12p40p70 and IL-6 and that Egmuc-treated DCs may activate NK cells, as judged by an increased expression of CD69. This evidence may contribute to the design of tumor vaccines and open new horizons in the use of parasite-derived molecules in the fight against cancer.
Subject(s)
Antineoplastic Agents/administration & dosage , Mucins/administration & dosage , Neoplasms/drug therapy , Peptides/administration & dosage , Animals , Antigens, CD/immunology , Antigens, Differentiation, T-Lymphocyte/immunology , Antineoplastic Agents/chemistry , Antineoplastic Agents/isolation & purification , Cancer Vaccines/immunology , Dendritic Cells/immunology , Echinococcus granulosus/chemistry , Humans , Interleukin-12 Subunit p40/immunology , Interleukin-6/immunology , Killer Cells, Natural/drug effects , Lectins, C-Type/immunology , Lymphocyte Activation/drug effects , Mice , Mucins/chemistry , Mucins/isolation & purification , Neoplasms/immunology , Neoplasms/pathology , Peptides/chemistry , Peptides/isolation & purification , Spleen/drug effects , Spleen/immunologyABSTRACT
CIGB-552 is a twenty-amino-acid novel synthetic peptide that has proven to be effective in reducing tumor size and increasing lifespan in tumor-bearing mice. Such capability is conferred by its cell-penetrating peptide character, which allows it to enter cells and elicit a pro-apoptotic effect through its major mediator, COMMD1 protein. Cell-penetrating peptides are able to use different internalization mechanisms, such as endocytosis or direct transduction through the plasma membrane. Although CIGB-552 cytotoxicity has been evaluated in several non-tumor- and tumor-derived cell lines, no data regarding the relationship between cell line sensitivity, cell penetrating capacity, the internalization mechanisms involved, COMMD1 expression levels, or its subcellular localization has yet been produced. Here, we present the results obtained from a comparative analysis of CIGB-552 sensitivity, internalization capacity and the mechanisms involved in three human tumor-derived cell lines from different origins: mammary gland, colon and lung (MCF-7, HT-29 and H460, respectively). Furthermore, cell surface markers relevant for internalization processes such as phosphatidylserine, as well as CIGB-552 target COMMD1 expression/localization, were also evaluated. We found that both endocytosis and transduction are involved in CIGB-552 internalization in the three cell lines evaluated. However, CIGB-552 incorporation efficiency and contribution of each mechanism is cell-line dependent. Finally, sensitivity was directly correlated with high internalization capacity in those cell lines where endocytosis had a major contribution on CIGB-552 internalization(AU)
Subject(s)
Humans , Antineoplastic Agents , Cell-Penetrating Peptides , Bibliography, National , UruguayABSTRACT
Because of resistance development by cancer cells against current anticancer drugs, there is a considerable interest in developing novel antitumor agents. We have previously demonstrated that CIGB-552, a novel cell-penetrating synthetic peptide, was effective in reducing tumor size and increasing lifespan in tumor-bearing mice. Studies of protein-peptide interactions have shown that COMMD1 protein is a major mediator of CIGB-552 antitumor activity. Furthermore, a typical serine-protease degradation pattern for CIGB-552 in BALB/c mice serum was identified, yielding peptides which differ from CIGB-552 in size and physical properties. In the present study, we show the results obtained from a comparative analysis between CIGB-552 and its main metabolites regarding physicochemical properties, cellular internalization, and their capability to elicit apoptosis in MCF-7 cells. None of the analyzed metabolites proved to be as effective as CIGB-552 in promoting apoptosis in MCF-7. Taking into account these results, it seemed important to examine their cell-penetrating capacity and interaction with COMMD1. We show that internalization, a lipid binding-dependent process, is impaired as well as metabolite-COMMD1 interaction, key component of the apoptotic mechanism. Altogether, our results suggest that features conferred by the amino acid sequence are decisive for CIGB-552 biological activity, turning it into the minimal functional unit(AU)
Subject(s)
Antineoplastic Agents/therapeutic use , Amino Acids , Apoptosis , Bibliography, National , UruguayABSTRACT
There is substantial evidence suggesting that certain parasites can have antitumor properties. We evaluated mucin peptides derived from the helminth Echinococcus granulosus (denominated Egmuc) as potential inducers of antitumor activity. We present data showing that Egmuc peptides were capable of inducing an increase of activated NK cells in the spleen of immunized mice, a fact that was correlated with the capacity of splenocytes to mediate killing of tumor cells. We demonstrated that Egmuc peptides enhance LPS-induced maturation of dendritic cells in vitro by increasing the production of IL-12p40p70 and IL-6 and that Egmuc-treated DCs may activate NK cells, as judged by an increased expression of CD69. This evidence may contribute to the design of tumor vaccines and open new horizons in the use of parasite-derived molecules in the fight against cancer(AU)