RESUMO
Actinoporins (APs) are soluble pore-forming proteins secreted by sea anemones that experience conformational changes originating in pores in the membranes that can lead to cell death. The processes involved in the binding and pore-formation of members of this protein family have been deeply examined in recent years; however, the intracellular responses to APs are only beginning to be understood. Unlike pore formers of bacterial origin, whose intracellular impact has been studied in more detail, currently, we only have knowledge of a few poorly integrated elements of the APs' intracellular action. In this review, we present and discuss an updated landscape of the studies aimed at understanding the intracellular pathways triggered in response to APs attack with particular reference to sticholysin II, the most active isoform produced by the Caribbean Sea anemone Stichodactyla helianthus. To achieve this, we first describe the major alterations these cytolysins elicit on simpler cells, such as non-nucleated mammalian erythrocytes, and then onto more complex eukaryotic cells, including tumor cells. This understanding has provided the basis for the development of novel applications of sticholysins such as the construction of immunotoxins directed against undesirable cells, such as tumor cells, and the design of a cancer vaccine platform. These are among the most interesting potential uses for the members of this toxin family that have been carried out in our laboratory.
Assuntos
Morte Celular/efeitos dos fármacos , Venenos de Cnidários/metabolismo , Venenos de Cnidários/toxicidade , Imunotoxinas/química , Imunotoxinas/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Anêmonas-do-Mar/química , AnimaisRESUMO
OBJECTIVES: Epithelial growth factor receptor (EGFR), as a malignancy marker, is overly expressed in multiple solid tumors including colorectal neoplasms, one of the most prevalent malignancies worldwide. The main objective of this study is to enhance the efficacy of anti-tumor therapy targeting EGFR by constructing a novel EGFR-specific immunotoxin (C-CUS245C) based on Cetuximab and recombinant Cucurmosin (CUS245C). METHODS: E. coli BL21 (DE3) PlysS (E. coli) was used to express CUS245C with a cysteine residue inserting to the C-terminus of Cucurmosin. Then immobilized metal ion affinity chromatography (IMAC) was used to purify CUS245C. The chemical conjugation method was used for the preparation of C-CUS245C. Then dialysis and IMAC were used to purify C-CUS245C. Western blot as well as SDS-PAGE was carried out to characterize the formation of C-CUS245C. At last the anti-colorectal cancer activity of C-CUS245C was investigated in vitro and in vivo. RESULTS: CUS245C with high purity could be obtained from the prokaryotic system. C-CUS245C was successfully constructed and highly purified. The cytotoxicity assays in vitro showed a significant proliferation inhibition of C-CUS245C on EGFR-positive cells for 120 h with IC50 values less than 0.1 pM. Besides, the anti-tumor efficacy of C-CUS245C was remarkably more potent than that of Cetuximab, CUS245C, and C + CUS245C (P < 0.001). Whereas the cytotoxicity of C-CUS245C could hardly be detected on EGFR-null cell line. Our results also showed that C-CUS245C had efficacy of anti-colorectal cancer in mouse xenograft model, indicating the therapeutic potential of C-CUS245C for the targeted therapy of colorectal neoplasms. CONCLUSIONS: C-CUS245C exhibits potent and EGFR-specific cytotoxicity. Insertional mutagenesis technique is worthy to be adopted in the preparation of immunotoxin. Immunotoxin can be highly purified through dialysis followed by IMAC.
Assuntos
Cetuximab/uso terapêutico , Neoplasias Colorretais/terapia , Imunotoxinas/uso terapêutico , Terapia de Alvo Molecular/métodos , Proteínas de Plantas/uso terapêutico , Animais , Antineoplásicos Imunológicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cetuximab/farmacologia , Cromatografia de Afinidade/métodos , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Escherichia coli/metabolismo , Humanos , Imunoconjugados/química , Imunoconjugados/uso terapêutico , Imunotoxinas/química , Imunotoxinas/isolamento & purificação , Imunotoxinas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Mutagênese Insercional/métodos , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Fatores de Tempo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The stage-specific embryonic antigen-4 (SSEA-4) is a cell surface glycosphingolipid antigen expressed in early stages of human development. This surface marker is downregulated during the differentiation process but is found re-expressed in several types of tumors, including breast cancer. This feature makes SSEA-4 an attractive target for the development of therapeutic antibodies against tumors. In this work, we first studied the binding and intracellular fate of the monoclonal antibody MC-813-70 directed against SSEA-4. MC-813-70 was found to be rapidly internalized into triple-negative breast cancer cells following binding to its target at the plasma membrane, and to accumulate in acidic organelles, most likely lysosomes. Given the internalization feature of MC-813-70, we next tested whether the antibody was able to selectively deliver the saporin toxin inside SSEA-4-expressing cells. Results show that the immunotoxin complex was properly endocytosed and able to reduce cell viability of breast cancer cells in vitro, either alone or in combination with chemotherapeutic drugs. Our findings indicate that the MC-813-70 antibody has the potential to be developed as an alternative targeted therapeutic agent for cancer cells expressing the SSEA-4 glycolipid.
Assuntos
Imunotoxinas/farmacologia , Saporinas/farmacologia , Antígenos Embrionários Estágio-Específicos/imunologia , Neoplasias de Mama Triplo Negativas/imunologia , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/imunologia , Adenocarcinoma/metabolismo , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Feminino , Humanos , Imunotoxinas/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Saporinas/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/metabolismoRESUMO
The toxicity of the curcin on cancer cells allows to consider this protein as the toxic component of an immunotoxin directed to Her2, which is associated with cancer. Reductive amination was proposed to conjugate curcin and an anti-Her2; the binding was tested using Polyacrylamide gel electrophoresis, western blot, and immunocytochemistry. The in vitro cytotoxicity of curcin and the immunotoxin was assessed on breast cancer cell lines SK-BR-3 (Her2(+)) and MDA-MB-231 (Her2(-)). IC50 values for curcin were 15.5 ± 8.3 and 18.6 ± 2.4 µg/mL, respectively, statistically equivalent (p < 0.05). While to the immunotoxin was 2.2 ± 0.08 for SK-BR-3 and 147.6 ± 2.5 µg/mL for MDA-MB-231. These values showed that the immunotoxin was seven times more toxic to the SK-BR-3 than curcin and eight times less toxic to the MDA-MB-231. The immunotoxin composed of curcin and an antibody against Her2 and constructed by reductive amination could be a therapeutic candidate against Her2(+) cancer.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Imunotoxinas/metabolismo , Receptor ErbB-2/imunologia , Proteínas Inativadoras de Ribossomos Tipo 1/metabolismo , Aminação , Linhagem Celular Tumoral , Fenômenos Químicos , Simulação por Computador , Humanos , Imunotoxinas/química , Imunotoxinas/imunologia , Modelos Moleculares , Oxirredução , Conformação Proteica , Proteínas Inativadoras de Ribossomos Tipo 1/químicaRESUMO
Gangliosides are sialic acid-containing glycolipids expressed on plasma membranes from nearly all vertebrate cells. The expression of ganglioside GD3, which plays essential roles in normal brain development, decreases in adults but is up regulated in neuroectodermal and epithelial derived cancers. R24 antibody, directed against ganglioside GD3, is a validated tumor target which is specifically endocytosed and accumulated in endosomes. Here, we exploit the internalization feature of the R24 antibody for the selective delivery of saporin, a ribosome-inactivating protein, to GD3-expressing cells [human (SK-Mel-28) and mouse (B16) melanoma cells and Chinese hamster ovary (CHO)-K1 cells]. This immunotoxin showed a specific cytotoxicity on tumor cells grew on 2D monolayers, which was further evident by the lack of any effect on GD3-negative cells. To estimate the potential antitumor activity of R24-saporin complex, we also evaluated the effect of the immunotoxin on the clonogenic growth of SK-Mel-28 and CHO-K1(GD3+) cells cultured in attachment-free conditions. A drastic growth inhibition (>80-90%) of the cell colonies was reached after 3 days of immunotoxin treatment. By the contrary, colonies continue to growth at the same concentration of the immuntoxin, but in the absence of R24 antibody, or in the absence of both immunotoxin and R24, undoubtedly indicating the specificity of the effect observed. Thus, the ganglioside GD3 emerge as a novel and attractive class of cell surface molecule for targeted delivery of cytotoxic agents and, therefore, provides a rationale for future therapeutic intervention in cancer.
Assuntos
Anticorpos/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Gangliosídeos/metabolismo , Imunotoxinas/metabolismo , Proteínas Inativadoras de Ribossomos Tipo 1/metabolismo , Animais , Células CHO , Proliferação de Células , Cricetinae , Cricetulus , Endossomos/metabolismo , Gangliosídeos/imunologia , Humanos , Imunotoxinas/farmacocinética , Camundongos , Microscopia Confocal , Microscopia de Fluorescência , Proteínas Inativadoras de Ribossomos Tipo 1/farmacocinética , Saporinas , Sais de Tetrazólio , TiazóisRESUMO
The review purposes are to (1) evaluate the experimental evidence for adverse effects on reproduction and metabolism and (2) identify the current knowledge of analytical procedures, biochemistry and environmental aspects relating to organotins. Organotins are pollutants that are used as biocides in antifouling paints. They produce endocrine-disrupting effects in mollusks, such as imposex. In rodents, organotin exposure induces developmental and reproductive toxicity as well as alteration of metabolic homeostasis through its action as an obesogen. The adverse effects that appear in rodents have raised concerns about organotins' potential health risk to humans in relation to organotin exposure. At present, triorganotin, such as tributyltin, have been demonstrated to produce imposex, and mammalian reproductive and metabolic toxicity. For most mammals, triorganotin exposure predominantly occurs through the ingestion, and this compound can cross the placenta. With these risks in mind, it is important to improve our knowledge of organotins' effects on environmental health.
Assuntos
Desinfetantes/toxicidade , Disruptores Endócrinos/toxicidade , Infertilidade Feminina/induzido quimicamente , Infertilidade Masculina/induzido quimicamente , Compostos Orgânicos de Estanho/toxicidade , Animais , Desinfetantes/química , Desinfetantes/metabolismo , Desinfetantes/farmacologia , Disruptores Endócrinos/química , Disruptores Endócrinos/metabolismo , Disruptores Endócrinos/farmacologia , Sistema Endócrino/efeitos dos fármacos , Sistema Endócrino/metabolismo , Metabolismo Energético/efeitos dos fármacos , Exposição Ambiental , Feminino , Humanos , Imunotoxinas/química , Imunotoxinas/metabolismo , Imunotoxinas/farmacologia , Imunotoxinas/toxicidade , Infertilidade Feminina/metabolismo , Infertilidade Masculina/metabolismo , Masculino , Obesidade/induzido quimicamente , Obesidade/metabolismo , Compostos Orgânicos de Estanho/química , Compostos Orgânicos de Estanho/metabolismo , Compostos Orgânicos de Estanho/farmacologia , Compostos de Trialquitina/química , Compostos de Trialquitina/metabolismo , Compostos de Trialquitina/farmacologia , Compostos de Trialquitina/toxicidadeRESUMO
BACKGROUND: Mercury (Hg) is a ubiquitous environmental contaminant with neurodevelopmental and immune system effects. An informative biomarker of Hg-induced immunotoxicity could aid studies on the potential contribution to immune-related health effects. OBJECTIVES: Our objectives were to test the hypothesis that methylmercury (MeHg) exposures affect levels of serum biomarkers and to examine interactions between Hg and selenium (Se) in terms of these responses. METHODS: This cross-sectional epidemiological study assessed adults living along the Tapajós River, a system long affected by MeHg. We measured antinuclear (ANA) and antinucleolar (ANoA) autoantibody levels and eight cytokines in serum samples (n = 232). Total Hg (including MeHg) and Se were measured in blood, plasma, hair, and urine. RESULTS: The median (range) total Hg concentrations were 14.1 µg/g (1.1-62.4), 53.5 µg/L (4.3-288.9), 8.8 µg/L (0.2-40), and 3.0 µg/L (0.2-16.1) for hair, blood, plasma, and urine, respectively. Elevated titers of ANA (but not ANoA) were positively associated with MeHg exposure (log-transformed, for blood and plasma), unadjusted [odds ratio (OR) = 2.6; 95% confidence interval (CI): 1.1, 6.2] and adjusted for sex and age (OR = 2.9; 95% CI: 1.1, 7.5). Proinflammatory [interleukin (IL)-6 and interferon (IFN)-γ], anti-inflammatory (IL-4), and IL-17 cytokine levels were increased with MeHg exposure; however, in the subset of the population with elevated ANA, proinflammatory IL-1ß, IL-6, IFN-γ, and tumor necrosis factor (TNF)-α and anti-inflammatory (IL-4) cytokine levels were decreased with MeHg exposure. Although Se status was associated with MeHg level (correlation coefficient = 0.86; 95% CI: 0.29, 1.43), Se status was not associated with any changes in ANA and did not modify associations between Hg and ANA titers. CONCLUSIONS: MeHg exposure was associated with an increased ANA and changes in serum cytokine profile. Moreover, alterations in serum cytokine profiles differed based on ANA response, suggesting a specific phenotype of MeHg susceptibility. Further research on the potential health implications of these observed immunological changes is warranted.
Assuntos
Biomarcadores/sangue , Exposição Ambiental , Poluentes Ambientais/metabolismo , Peixes/metabolismo , Imunotoxinas/metabolismo , Compostos de Metilmercúrio/metabolismo , Selênio/metabolismo , Animais , Anticorpos Antinucleares/sangue , Autoanticorpos/sangue , Brasil , Estudos Transversais , Citocinas/sangue , Poluentes Ambientais/sangue , Poluentes Ambientais/urina , Humanos , Imunotoxinas/sangue , Imunotoxinas/urina , Compostos de Metilmercúrio/sangue , Compostos de Metilmercúrio/urina , Razão de ChancesRESUMO
The use of pore-forming toxins from sea anemones (actinoporins) in the construction of immunotoxins (ITs) against tumour cells is an alternative for cancer therapy. However, the main disadvantage of actinoporin-based ITs obtained so far has been the poor cellular specificity associated with the toxin's ability to bind and exert its activity in almost any cell membrane. Our final goal is the construction of tumour proteinase-activated ITs using a cysteine mutant at the membrane binding region of sticholysin-I (StI), a cytolysin isolated from the sea anemone Stichodactyla helianthus. The mutant and the ligand moiety would be linked by proteinase-sensitive peptides through the StI cysteine residue blocking the toxin binding region and hence the IT non-specific killing activity. To accomplish this objective the first step was to obtain the mutant StI W111C, and to evaluate the impact of mutating tryptophan 111 by cysteine on the toxin pore-forming capacity. After proteolysis of the cleavage sequence, a short peptide would remain attached to the toxin. The next step was to evaluate whether this mutant is able to form pores even with a residual peptide linked to cysteine 111. In this work we demonstrated that (i) StI W111C shows pore-forming capacity in a nanomolar range, although it is 8-fold less active than the wild-type recombinant StI, corroborating the previously reported importance of residue 111 for the binding of StI to membranes, and (ii) the mutant is able to form pores even with a residual seven-residue peptide linked to cysteine 111. In addition, it was demonstrated that binding of a large molecule to cysteine 111 renders an inactive toxin that is no longer able to bind to the membrane. These results validate the mutant StI W111C for its use in the construction of tumour proteinase-activated ITs.
Assuntos
Imunotoxinas/química , Proteínas Citotóxicas Formadoras de Poros/química , Animais , Membrana Celular/química , Membrana Celular/metabolismo , Cromatografia em Gel , Cromatografia por Troca Iônica , Dimerização , Imunotoxinas/genética , Imunotoxinas/isolamento & purificação , Imunotoxinas/metabolismo , Modelos Moleculares , Mutação , Compostos Orgânicos/química , Compostos Orgânicos/isolamento & purificação , Compostos Orgânicos/metabolismo , Perforina , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/isolamento & purificação , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Ligação Proteica , Reprodutibilidade dos Testes , Anêmonas-do-MarRESUMO
The use of membrane active toxins as toxic moieties in the construction of immunotoxins (ITs) is an attractive alternative to overcome some of the problems of classical ITs since these new conjugates are based in the use of a different mechanism of killing undesired cells. Pore-forming cytolysins from sea anemones were used in the construction of ITs targeted to different cell types including tumour cell lines and the parasite Giardia duodenalis. The results obtained support the feasibility of directing these cytolysins to the surface of the cancer cells or the parasite through their conjugation to monoclonal antibodies recognizing tumour-associated or parasite antigens, respectively. However the main problem with the IT constructed in this fashion is the lack of specificity associated with the toxin moiety. An approach designed to overcome this limitation was the construction of inactive cytolysin with built-in biological "trigger" that renders the toxin active in the presence of tumour-specific proteinases. This construction is considered as a proof of concept to demonstrate the feasibility of such activation systems in the construction of ITs based on pore-forming cytolysins from sea anemones with reduced unspecific activity. The future prospects of the use of the N-terminal region of actinoporins for construction of IT is also described.
Assuntos
Citotoxinas/toxicidade , Imunotoxinas/química , Imunotoxinas/toxicidade , Anêmonas-do-Mar/química , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/imunologia , Citotoxinas/química , Giardia/efeitos dos fármacos , Giardia/crescimento & desenvolvimento , Giardia/imunologia , Humanos , Imunotoxinas/imunologia , Imunotoxinas/metabolismoRESUMO
Las células dendríticas epidérmicas Thy-1 positivas (CDE Thy-1) son un tipo celular recientemente descubierto en la epidermis de ratones. Estás células se caracterizan por presentar en su superficie el antígeno Thy-1, que hasta 1975 se había encontrado sólo en timocitos y células nerviosas. Las CDE Thy-1 expresan las glicoproteínas CD3 y gamma delta (* *) del receptor antígenico de linfocitos T, lo cual las relaciona consistentemente con este grupo celular. Hasta el presente se desconoce la función específica de este nuevo tipo celular. Sin embargo, experimentos in vitro han demostrado que estas células presentan citotoxicidad frente a los grupos de células tumorales. Por otra parte, pruebas in vivo demuestran que las CDE Thy-1 ejercen una función derreguladora en las repuestas de hipersensibilidad por contacto. Además su ubicación en el epitelio y la expresión del receptor antigénico **, han permitido asociarlas a mecanismos de vigilancia inmunológica de la integridad cutánea a través del reconocimiento antigénico de proteínas de stress