RESUMO

Amblyomin-X is a Kunitz-type FXa inhibitor identified through the transcriptome analysis of the salivary gland from Amblyomma sculptum tick. This protein consists of two domains of equivalent size, triggers apoptosis in different tumor cell lines, and promotes regression of tumor growth, and reduction of metastasis. To study the structural properties and functional roles of the N-terminal (N-ter) and C-terminal (C-ter) domains of Amblyomin-X, we synthesized them by solid-phase peptide synthesis, solved the X-Ray crystallographic structure of the N-ter domain, confirming its Kunitz-type signature, and studied their biological properties. We show here that the C-ter domain is responsible for the uptake of Amblyomin-X by tumor cells and highlight the ability of this domain to deliver intracellular cargo by the strong enhancement of the intracellular detection of molecules with low cellular-uptake efficiency (p15) after their coupling with the C-ter domain. In contrast, the N-ter Kunitz domain of Amblyomin-X is not capable of crossing through the cell membrane but is associated with tumor cell cytotoxicity when it is microinjected into the cells or fused to TAT cell-penetrating peptide. Additionally, we identify the minimum length C-terminal domain named F2C able to enter in the SK-MEL-28 cells and induces dynein chains gene expression modulation, a molecular motor that plays a role in the uptake and intracellular trafficking of Amblyomin-X.

RESUMO

Amblyomin-X is a Kunitz-type FXa inhibitor identified through the transcriptome analysis of the salivary gland from Amblyomma sculptum tick. This protein consists of two domains of equivalent size, triggers apoptosis in different tumor cell lines, and promotes regression of tumor growth, and reduction of metastasis. To study the structural properties and functional roles of the N-terminal (N-ter) and C-terminal (C-ter) domains of Amblyomin-X, we synthesized them by solid-phase peptide synthesis, solved the X-Ray crystallographic structure of the N-ter domain, confirming its Kunitz-type signature, and studied their biological properties. We show here that the C-ter domain is responsible for the uptake of Amblyomin-X by tumor cells and highlight the ability of this domain to deliver intracellular cargo by the strong enhancement of the intracellular detection of molecules with low cellular-uptake efficiency (p15) after their coupling with the C-ter domain. In contrast, the N-ter Kunitz domain of Amblyomin-X is not capable of crossing through the cell membrane but is associated with tumor cell cytotoxicity when it is microinjected into the cells or fused to TAT cell-penetrating peptide. Additionally, we identify the minimum length C-terminal domain named F2C able to enter in the SK-MEL-28 cells and induces dynein chains gene expression modulation, a molecular motor that plays a role in the uptake and intracellular trafficking of Amblyomin-X.

RESUMO

Salivary glands are vital structures responsible for successful tick feeding. The saliva of ticks contains numerous active molecules that participate in several physiological processes. A Kunitz-type factor Xa (FXa) inhibitor, similar to the tissue factor pathway inhibitor (TFPI) precursor, was identified in the salivary gland transcriptome of Amblyomma sculptum ticks. The recombinant mature form of this Kunitz-type inhibitor, named Amblyomin-X, displayed anticoagulant, antiangiogenic, and antitumor properties. Amblyomin-X is a protein that inhibits FXa in the blood coagulation cascade and acts via non-hemostatic mechanisms, such as proteasome inhibition. Amblyomin-X selectively induces apoptosis in cancer cells and promotes tumor regression through these mechanisms. Notably, the cytotoxicity of Amblyomin-X seems to be restricted to tumor cells and does not affect non-tumorigenic cells, tissues, and organs, making this recombinant protein an attractive molecule for anticancer therapy. The cytotoxic activity of Amblyomin-X on tumor cells has led to vast exploration into this protein. Here, we summarize the function, action mechanisms, structural features, pharmacokinetics, and biodistribution of this tick Kunitz-type inhibitor recombinant protein as a promising novel antitumor drug candidate.

RESUMO

Salivary glands are vital structures responsible for successful tick feeding. The saliva of ticks contains numerous active molecules that participate in several physiological processes. A Kunitz-type factor Xa (FXa) inhibitor, similar to the tissue factor pathway inhibitor (TFPI) precursor, was identified in the salivary gland transcriptome of Amblyomma sculptum ticks. The recombinant mature form of this Kunitz-type inhibitor, named Amblyomin-X, displayed anticoagulant, antiangiogenic, and antitumor properties. Amblyomin-X is a protein that inhibits FXa in the blood coagulation cascade and acts via non-hemostatic mechanisms, such as proteasome inhibition. Amblyomin-X selectively induces apoptosis in cancer cells and promotes tumor regression through these mechanisms. Notably, the cytotoxicity of Amblyomin-X seems to be restricted to tumor cells and does not affect non-tumorigenic cells, tissues, and organs, making this recombinant protein an attractive molecule for anticancer therapy. The cytotoxic activity of Amblyomin-X on tumor cells has led to vast exploration into this protein. Here, we summarize the function, action mechanisms, structural features, pharmacokinetics, and biodistribution of this tick Kunitz-type inhibitor recombinant protein as a promising novel antitumor drug candidate.

Assuntos

RESUMO

Dystrophin deficiency makes the sarcolemma fragile and susceptible to degeneration in Duchenne muscular dystrophy. The proteasome is a multimeric protease complex and is central to the regulation of cellular proteins. Previous studies have shown that proteasome inhibition improved pathological changes in mdx mice. Ixazomib is the first oral proteasome inhibitor used as a therapy in multiple myeloma. This study investigated the effects of ixazomib on the dystrophic muscle of mdx mice. MDX mice were treated with ixazomib (7.5 mg/kg/wk by gavage) or 0.2 mL of saline for 12 weeks. The Kondziela test was performed to measure muscle strength. The tibialis anterior (TA) and diaphragm (DIA) muscles were used for morphological analysis, and blood samples were collected for biochemical measurement. We observed maintenance of the muscle strength in the animals treated with ixazomib. Treatment with ixazomib had no toxic effect on the mdx mouse. The morphological analysis showed a reduction in the inflammatory area and fibres with central nuclei in the TA and DIA muscles and an increase in the number of fibres with a diameter of 20 µm2 in the DIA muscle after treatment with ixazomib. There was an increase in the expression of dystrophin and utrophin in the TA and DIA muscles and a reduction in the expression of osteopontin and TGF-ß in the DIA muscle of mdx mice treated with ixazomib. Ixazomib was thus shown to increase the expression of dystrophin and utrophin associated with improved pathological and functional changes in the dystrophic muscles of mdx mice.

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Bortezomib, an inhibitor of 26S proteasome, is an anti-cancer therapeutic agent used in different cancer types. It leads to the arrest of the cancerous cell cycle by inhibiting angiogenesis and inducing apoptosis. Liver is the vital organ for detoxification and excretion of toxic products. The treatment with chemotherapy is a challenge, drugs are used to destroy cancer cells, but healthy cells can be affected during cancer treatment as well. The main objective of this study was to analyze the histopathological and biochemical effects of bortezomib on liver. Twenty-four female C57BL/6 mice were distributed into 4 groups, bortezomib injected treatment groups (Btz1, Btz2) and saline injected control groups (C1, C2). Bortezomib and saline were treated twice per week for 6 weeks and sacrificed at the end of one day (Btz1, C1) and 4 weeks (Btz2, C2) after the last injection. Liver samples were examined for histopathological analysis and the serum samples processed for biochemical analysis. Tissue samples were fixed, routinely processed, sectioned, and stained with Hematoxylin and Eosin (H&E). Periodic Acid-Schiff (PAS), Sudan Black staining and Masson's trichrome histochemical staining methods were performed to characterize the lesions. Histopathological analysis of the Btz1 and Btz2 groups revealed acute hepatic morphological changes such as hepatocellular swelling (cloudy swelling), necro-inflammatory reaction, and increased mononuclear polyploidy. Based on the negative staining with PAS and Sudan Black staining, hepatocellular swelling was diagnosed as hydropic degeneration. Necro-inflammatory reaction observed in the form of acute hepatitis was composed of mainly mononuclear cell infiltration accompanied by multifocal necrotic foci. Kupffer cell proliferation was observed in parallel with degenerative and necrotic changes. An Increase in hepatocellular mononuclear polyploidy visualized as hepatocytes with a single enlarged nucleus was detected in all liver sections of Btz1 and Btz2 groups. Individual cases of cholestasis (n = 1) and mild hepatic fibrosis (n = 1) were also reported. Significant elevated levels of alanine aminotransferase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) and gamma-glutamyl transferase (GGT) were detected in bortezomib treated groups. Few clinical cases reported liver injury related to bortezomib used for cancer treatment. However, the liver was not considered as a target for bortezomib treatment. Our data suggesting that bortezomib caused liver damage and induces elevations in serum levels. The reported hepatic lesions including hepatocellular swelling, acute hepatitis and mononuclear polyploidy were mainly mild and moderate in severity. The increase of polyploidy in liver tissue of mice treated with bortezomib in this study was explained as a reaction of the liver facing the drug-induced hepatic damage. The mechanism leading to the hepatotoxicity of bortezomib treatment is not known but the production of a toxic metabolite through its metabolism in the liver can be suggested. Moreover, no recovery was also observed in histopathological and biochemical analyses suggesting that the bortezomib effect is non-reversible four weeks after the drug was withdrawn. Patients should be informed about the possibility of acute drug-induced hepatitis and hepatotoxicity of this chemotherapeutic agent after the treatment.(AU)

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Proteasome inhibitors such as carfilzomib are indicated in multiple myeloma patients showing relapse and/or refractoriness of clonal activity. However, this therapy has been associated with a significant incidence of cardiotoxicity, especially in patients with known cardiovascular risk factors. Here we report a case of a 60-year-old woman with multiple myeloma, who developed severe congestive heart failure with positive myocardial injury biomarkers together with impaired LVEF and GLS, after treatment with carfilzomib. Therefore, chemotherapeutic drug was discontinued and neurohormonal blocking and diuretic therapy was started resulting in amelioration of symptoms, without changes in LVEF but with significant GLS improvement. Although high-grade cardiotoxicity is relatively rare in patients with non previous cardiac risk factors, it was a predictable side effect of carfilzomib use. Recognition of this syndrome is critical to instauration of appropriate therapy and prevention of morbimortality.

RESUMO

The aim of this study was to describe clinical and survival characteristics of transplant-eligible multiple myeloma (MM) patients in Latin America (LA), with a special focus on differences between public and private healthcare facilities. We included 1293 patients diagnosed between 2010 and 2018. A great disparity in outcomes and survival between both groups was observed. Late diagnosis and low access to adequate frontline therapy and ASCT in public institutions probably explain these differences. Patients treated with novel drug induction protocols, followed by autologous stem cell transplantation (ASCT) and maintenance, have similar overall survival compared to that published internationally.

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BACKGROUND: Nuclear factor κB (NF-κB) comprises a family of proteins that act as transcription factors promoting the expression of many genes. Activation of NF-κB biochemical cascades is associated with the regulation of innate and adaptive immune responses and inflammation, among other physiological responses. However, genetic abnormalities and continuous stimulation of the NF- κB-IKKß pathway are directly related to many types of inflammatory and autoimmune diseases, as well as to the genesis and survival of tumor cells. OBJECTIVES: Inhibition of the NF-κB-IKKß cascade can be considered an attractive therapeutic method for the genesis of new prototypes to combat these chronic multifactorial diseases. RESULTS: This review describes some prototypes and drugs that act to inhibit the NF-κB-IKKß pathway, highlighting the realities, challenges and perspectives for therapeutic use. CONCLUSION: Although only proteasome inhibitors, such as bortezomib and carfilzomib, are a reality as therapeutically useful drugs among the known modulators of possible targets in the NF-κB-IKKß pathway, some other prototypes described as IKKß inhibitors have entered clinical stages as drug candidates for the control of inflammatory diseases. It is important to note that some classical drugs available on the pharmaceutical market, such as acetylsalicylic acid, were also described more recently as NF-κB pathway modulators as IKKß inhibitors.

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We report a case of capillary leak that developed during treatment of antibody-mediated rejection in a kidney transplant recipient. A 53-year-old female transplant recipient experienced an increase in serum creatinine from 1.1 to 1.8 mg/dL. Antibody-mediated rejection was diagnosed by graft biopsy. She was treated with five plasmapheresis sessions (on alternate days with albumin replacement), five doses of immunoglobulin (5 g/dose at 100 mg/kg), a single dose of rituximab (500 mg), and four doses of bortezomib on days 1, 4, 7, and 10 (1.72 mg/dose at 1.3 mg/m2 body surface area). During treatment, edema, slight diarrhea, pancytopenia, hypoalbuminemia, peripheral neuropathy, and postural hypotension were noted. Despite control of liquids, she presented with edema progressing to an increase of more than 10 kg body weight. Prerenal acute graft dysfunction associated with hypotension was diagnosed on day 12, heart failure or other infectious complications being discounted. On day 13, daily hemodialysis was prescribed, and a stable volume status was reached after five hemodialysis sessions. On day 20, the patient recovered diuresis and the edema and diarrhea abated, but she remained on chronic hemodialysis. After excluding other causes of distributive shock, the diagnosis of capillary leak syndrome was based on the presence of hypotension, generalized edema, and hypoalbuminemia in the absence of significant proteinuria. The concomitant presence of diarrhea, peripheral neuropathy, and pancytopenia, suggest a possible causal role for bortezomib. Awareness by clinicians of capillary leak syndrome associated with bortezomib-based treatment of AMR is paramount, despite its rarity.

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BACKGROUND: Considerable evidence demonstrates the importance of dithiocarbamates especially disulfiram as anticancer drugs. However there are no systematic reviews outlining how their metal-binding ability is related to their anticancer activity. This review aims to summarize chemical features and metal-binding activity of disulfiram and its metabolite DEDTC, and discuss different mechanisms of action of disulfiram and their contributions to the drug's anticancer activity. METHODS: We undertook a disulfiram-related search on bibliographic databases of peerreviewed research literature, including many historic papers and in vitro, in vivo, preclinical and clinical studies. The selected papers were carefully reviewed and summarized. RESULTS: More than five hundreds of papers were obtained in the initial search and one hundred eighteen (118) papers were included in the review, most of which deal with chemical and biological aspects of Disulfiram and the relationship of its chemical and biological properties. Eighty one (81) papers outline biological aspects of dithiocarbamates, and fifty seven (57) papers report biological activity of Disulfiram as an inhibitor of proteasomes or inhibitor of aldehyde dehydrogenase enzymes, interaction with other anticancer drugs, or mechanism of action related to reactive oxygen species. Other papers reviewed focus on chemical aspects of dithiocarbamates. CONCLUSION: This review confirms the importance of chemical features of compounds such as Disulfiram to their biological activities, and supports repurposing DSF as a potential anticancer agent.

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Proteasomal proteolysis is required for a wide range of cellular processes, including protein quality control, cell cycle progression, cell death and metabolic adaptation to environment changes or stress responses. Proteasome inhibitors are useful compounds for determining the roles of proteasome in eukaryotic cells. Here, we investigated the effects of gliotoxin, a proteasome inhibitor, on the cell growth, replication, ultrastructure, DNA integrity and proteasomal proteolytic activity of the protist parasite Tritrichomonas foetus. The effect of gliotoxin on the transformation of T. foetus to endoflagellar form (EFF), also known as pseudocyst, was investigated. Gliotoxin inhibited the culture growth, arrested cell cycle, and provoked a trichomonacidal effect in a dose-dependent manner. Parasites treated with gliotoxin displayed features typical of cell death, such as membrane blebbing, concentric membrane whorls containing remnants of organelles, intense cytosolic and nuclear vacuolisation, chromatin condensation, DNA fragmentation, cytoplasmic disintegration and plasma membrane disruption. The proteasomal peptidase activity was inhibited by gliotoxin in a dose-dependent manner. Gliotoxin treatment also induced an irreversible EFF transformation in a dose/time-dependent manner. We compared morphological characteristics between gliotoxin- and cold-induced EFF parasites. Our results suggest that gliotoxin could induce EFF transformation by a mechanism distinct from that provoked by cold temperature. This study further contributes to a better understanding of the role of proteasome system in cell cycle, cell death and EFF transformation in T. foetus.

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During the last two decades, new insights into proteasome function and its role in several human diseases made it a potential therapeutic target. In this context, Amblyomin-X is a Kunitz-type FXa inhibitor similar to endogenous tissue factor pathway inhibitor (TFPI) and is a novel proteasome inhibitor. Herein, we have demonstrated Amblyomin-X cytotoxicity to different tumor cells lines such as pancreatic (Panc1, AsPC1BxPC3) and melanoma (SK-MEL-5 and SK-MEL-28). Of note, Amblyomin-X was not cytotoxic to normal human fibroblast cells. In addition, Amblyomin-X promoted accumulation of ER stress markers (GRP78 and GADD153) in sensitive (SK-MEL-28) and bortezomib-resistant (Mia-PaCa-2) tumor cells. The intracellular calcium concentration [Ca(2+)] i was slightly modulated in human tumor cells (SK-MEL-28 and Mia-PaCa-2) after 24 h of Amblyomin-X treatment. Furthermore, Amblyomin-X induced mitochondrial dysfunction, cytochrome-c release, PARP cleavage, and activation of caspase cascade in both human tumor (SK-MEL-28 and Mia-PaCa-2) cells. These investigations might help in further understanding of the antitumor properties of Amblyomin-X.

Assuntos