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1.
Ann Surg Oncol ; 2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33856603

RESUMO

BACKGROUND: Management of patients with sentinel lymph node (SLN)-positive melanoma has changed dramatically over the last few years such that completion lymph node dissection (CLND) has become uncommon, and many patients receive adjuvant immunotherapy or targeted therapy. This study seeks to characterize patterns and predictors of early recurrence in this setting. PATIENTS AND METHODS: All patients with primary cutaneous melanoma undergoing sentinel lymph node biopsy (SLNB) between 3/2016 and 12/2019 were identified. The subset with a positive SLN who did not undergo CLND were examined for further analysis of outcomes and predictors of recurrence. RESULTS: Overall, 215 patients with SLN-positive melanoma who did not have CLND were identified. Adjuvant systemic therapy was administered to 102 (47%), with 93% of this subset receiving immunotherapy (n = 95). Median follow-up from SLNB was 20 months (IQR 12-28.5 months), and 57 patients (27%) recurred during this time. The SLN basin was the most common site of recurrence (n = 38, 67% of recurrence), with isolated nodal recurrence being the most common first site of recurrent disease (n = 22, 39% of recurrence). On multivariable analysis, lymphovascular invasion (LVI) of the primary tumor, two or more involved nodes, and > 1 mm nodal deposit were independently associated with higher rates of nodal relapse. CONCLUSIONS: Nodal recurrence is a primary driver of early disease relapse for patients with SLN-positive melanoma who do not undergo CLND in the era of effective adjuvant systemic therapy. LVI, ≥ 2 nodes, or > 1 mm nodal disease identifies patients at particularly high risk of nodal relapse.

2.
Sci Adv ; 7(14)2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33789895

RESUMO

Intake of processed foods has increased markedly over the past decades, coinciding with increased microvascular diseases such as chronic kidney disease (CKD) and diabetes. Here, we show in rodent models that long-term consumption of a processed diet drives intestinal barrier permeability and an increased risk of CKD. Inhibition of the advanced glycation pathway, which generates Maillard reaction products within foods upon thermal processing, reversed kidney injury. Consequently, a processed diet leads to innate immune complement activation and local kidney inflammation and injury via the potent proinflammatory effector molecule complement 5a (C5a). In a mouse model of diabetes, a high resistant starch fiber diet maintained gut barrier integrity and decreased severity of kidney injury via suppression of complement. These results demonstrate mechanisms by which processed foods cause inflammation that leads to chronic disease.

3.
Colorectal Dis ; 2021 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-33793063

RESUMO

INTRODUCTION: The COVID-19 pandemic led to widespread disruption of colorectal cancer services during 2020. Established cancer referral pathways were modified in response to reduced diagnostic availability. The aim of this paper is to assess the impact of COVID-19 on colorectal cancer referral, presentation and stage. METHODS: This was a single centre, retrospective cohort study performed at a tertiary referral centre. Patients diagnosed and managed with colorectal adenocarcinoma between January to December 2020 were compared with patients from 2018 and 2019 in terms of demographics, mode of presentation and pathological cancer staging. RESULTS: 272 patients were diagnosed with colorectal adenocarcinoma during 2020 compared with 282 in 2019 and 257 in 2018. Patients in all years were comparable for age, gender, and tumour location (p=>0.05). There was a significant decrease in Urgent Suspected Cancer (USC) referrals, diagnostic colonoscopy and radiological imaging performed between March to June 2020 compared with previous years. More patients presented as emergencies (p=0.03) with increased rates of large bowel obstruction in 2020 compared with 2018/19 (p=0.01). The distribution of TNM grade was similar across the 3 years but more T4 cancers were diagnosed in 2020 versus 2018/19 (p=0.03). CONCLUSION: This study demonstrates that a relatively short-term impact on the colorectal cancer referral pathway can have significant consequences on patient presentation leading to higher risk emergency presentation and surgery at a more advanced stage. It is critical therefore that efforts are made to make this pathway more robust to minimise the impact of other future adverse events and to consolidate the benefits of earlier diagnosis and treatment.

4.
Oncologist ; 2021 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-33861486

RESUMO

LESSONS LEARNED: This study suggests that trametinib has significant clinical activity in non-V600 BRAF mutation and BRAF fusion metastatic melanoma, albeit in a small cohort. All patients with metastatic melanoma should undergo sequencing of the BRAF gene to identify noncanonical BRAF mutations that may indicate benefit from treatment with trametinib. BACKGROUND: Non-V600 BRAF mutations and BRAF fusions in aggregate occur in approximately 5% of all melanomas. Inhibition of the mitogen-activated protein kinase (MAPK) pathway has been implicated as a possible treatment strategy for these patients. METHODS: In this open-label, multicenter, phase II study, patients with advanced melanoma harboring mutations in BRAF outside V600 (non-V600) or BRAF fusions received trametinib 2.0 mg daily. Patients were divided into cohorts based on the intrinsic catalytic activity of BRAF mutation (high, cohort A; low/unknown, cohort B). The primary endpoint was objective response rate (ORR) for patients in cohort A; secondary endpoints included ORR in cohort B, safety, and survival in both treatment arms. RESULTS: Among all patients, the ORR was 33% (three of nine patients), including 67% in cohort A and 17% in cohort B. Two patients had stable disease as best response, and six patients had some degree of tumor shrinkage. The median progression-free survival (PFS) was 7.3 months. Treatment-related adverse events occurred in all patients (100%); most (89%) were grade 1-2. CONCLUSION: In contrast to recently described tumor-agnostic studies in a genetically similar population, trametinib had considerable activity in a small population of patients with melanoma harboring BRAF non-V600 mutations and fusions, providing rationale for sequencing in search of these genomic alterations.

5.
Free Radic Biol Med ; 169: 1-11, 2021 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-33819622

RESUMO

Humans have extensive adverse exposure to alpha,beta-unsaturated carbonyl compounds (ABuCs) as these are major toxins in smoke and exhaust fumes, as well as products of lipid peroxidation. In contrast, another ABuC, dimethylfumarate, is used to treat psoriasis and multiple sclerosis. ABuCs undergo Michael adduction with amine, imidazole and thiol groups, with reaction at Cys residues predominating. Here we report rate constants, k2, for ABuCs (acrolein, crotonaldehyde, dimethylfumarate, cyclohex-1-en-2-one, cyclopent-1-en-2-one) with Cys residues present on N-Ac-Cys, GSH, bovine serum albumin, creatine kinase, papain, glyceraldehyde-3-phosphate dehydrogenase, and both wild-type and the C151S mutant of Keap-1. k2 values for N-Ac-Cys and GSH vary by > 250-fold, indicating a marked ABuC structure dependence, with acrolein the most reactive. There is also considerable variation in k2 between protein Cys groups, with these significantly greater than for GSH. A linear inverse correlation for acrolein with the thiol pKa indicates that the thiolate anion is the reactive species. The modest k2 for GSH rationalizes the detection of protein adducts of ABuCs in cells. The k2 values for dimethylfumarate also vary markedly, with the Cys151 residue on Keap-1 being particularly reactive, with the C151S mutant giving a much lower k2 value. The data for crotonaldehyde, dimethylfumarate, and cyclohex-1-en-2-one show little correlation with the Cys pKa values, indicating that steric/electronic interactions, rather than Cys ionization are important. These data indicate that protein Cys residues, and particularly Cys151 on Keap-1, react readily with dimethylfumarate, and this may help rationalize the use of this compound as a therapeutic agent.

7.
Cancer Immunol Res ; 2021 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-33653716

RESUMO

Immunotherapy (IT) and targeted therapy (TT) are both effective against melanoma, but their combination is frequently toxic. Here, we investigated whether the sequence of IT (anti-PD-1)→ TT (ceritinib-trametinib or dabrafenib-trametinib) was associated with improved antitumor responses in mouse models of BRAF- and NRAS-mutant melanoma. Mice with NRAS-mutant (SW1) or BRAF-mutant (SM1) mouse melanomas were treated with either IT, TT, or the sequence of IT→TT. Tumor volumes were measured, and samples from the NRAS-mutant melanomas were collected for immune-cell analysis, single-cell RNA sequencing (scRNA-seq), and reverse phase protein analysis (RPPA). scRNA-seq demonstrated that the IT→TT sequence modulated the immune environment, leading to increased infiltration of T cells, monocytes, dendritic cells and natural killer cells, and decreased numbers of tumor-associated macrophages, myeloid-derived suppressor cells, and regulatory T cells. Durable responses to the IT→TT sequence were dependent on T-cell activity, with depletion of CD8+, but not CD4+, T cells abrogating the therapeutic response. An analysis of transcriptional heterogeneity in the melanoma compartment showed the sequence of IT→TT enriched for a population of melanoma cells with increased expression of MHC class I and melanoma antigens. RPPA analysis demonstrated that the sustained immune response induced by IT→TT suppressed tumor-intrinsic signaling pathways required for therapeutic escape. These studies establish that upfront IT improves the responses to TT in BRAF- and NRAS-mutant melanoma models.

8.
Redox Biol ; 41: 101925, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33714740

RESUMO

Inter- and intra-molecular crosslinks can generate protein dysfunction, and are associated with protein aggregate accumulation in aged and diseased tissues. Crosslinks formed between multiple amino acid side chains can be reversible or irreversible. Disulfides formed either enzymatically, or as a result of oxidant-mediated reactions, are a major class of reversible crosslinks. Whilst these are commonly generated via oxidation of Cys thiol groups, they are also formed by 'oxidant-mediated thiol-disulfide reactions' via initial disulfide oxidation to a thiosulfinate or zwitterionic peroxide, and subsequent reaction with another thiol including those on other proteins. This generates new intermolecular protein-protein crosslinks. Here we demonstrate that photooxidation, or reaction with the biological oxidants HOCl and ONOOH, of the single disulfide present in the major human plasma inflammatory protein, C-reactive protein (CRP) can give rise to reversible disulfide bond formation with human serum albumin (HSA). This occurs in an oxidant dose-, or illumination-time-, dependent manner. These CRP-HSA crosslinks are formed both in isolated protein systems, and in fresh human plasma samples containing high, but not low, levels of CRP. The inter-protein crosslinks which involve Cys36 of CRP and Cys34 of HSA, have been detected by both immunoblotting and mass spectrometry (MS). The yield of protein-protein crosslinks depends on the nature and extent of oxidant exposure, and can be reversed by dithiothreitol and tris(2-carboxyethyl)phosphine hydrochloride. These data indicate that oxidation of disulfide bonds in proteins can be a source of novel inter-protein crosslinks, which may help rationalize the accumulation of crosslinked proteins in aged and diseased tissues.

9.
Cancer Med ; 10(7): 2293-2299, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33686688

RESUMO

BACKGROUND: Anti-programmed cell death protein 1 (PD-1) antibodies are a standard treatment for metastatic melanoma patients. However, the understanding of the efficacy of anti-PD-1 for acral melanoma (AM) and mucosal melanoma (MM) is limited as these subtypes are relatively rare compared to cutaneous melanoma (CM). METHODS: This single institution, retrospective cohort study included patients with advanced AM and MM who underwent anti-PD-1 therapy for metastatic melanoma between 2012 and 2018. Objective responses were determined using the investigator-assessed Response Evaluation Criteria in Solid Tumors version 1.1. Progression-free survival (PFS) and overall survival (OS) were assessed using the Kaplan-Meier method. A Cox regression analysis was performed to identify the factors associated with survival outcomes. RESULTS: Ninety-seven patients were identified, 38 (39%) with AM and 59 (61%) with MM. The objective response rates (ORRs) were 21.0% and 15.2% in patients with AM and MM, respectively. The median PFS and OS were 3.6 and 25.7 months for AM patients, and 3.0 and 20.1 months for MM patients, respectively. Elevated serum lactate dehydrogenase (LDH) (AM: hazard ratio [HR], 0.22; 95% confidence interval [CI], 0.06-0.87; p = 0.03, MM: HR, 0.20; 95% CI, 0.08-0.53; p = 0.001) was significantly associated with shorter OS for both subtypes. CONCLUSIONS: The ORR, PFS, and OS with anti-PD-1 therapy were poor in patients with AM and MM compared to those previously reported clinical trials for nonacral CM. High serum LDH was associated with significantly shorter OS.

10.
Proc Natl Acad Sci U S A ; 118(13)2021 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-33766916

RESUMO

The freezing of water into ice is one of the most important processes in the physical sciences. However, it is still not understood at the molecular level. In particular, the crystallization of cubic ice ([Formula: see text])-rather than the traditional hexagonal polytype ([Formula: see text])-has become an increasingly debated topic. Although evidence for [Formula: see text] is thought to date back almost 400 y, it is only in the last year that pure [Formula: see text] has been made in the laboratory, and these processes involved high-pressure ice phases. Since this demonstrates that pure [Formula: see text] can form, the question naturally arises if [Formula: see text] can be made from liquid water. With this in mind, we have performed a high-throughput computational screening study involving molecular dynamics simulations of nucleation on over 1,100 model substrates. From these simulations, we find that 1) many different substrates can promote the formation of pristine [Formula: see text]; 2) [Formula: see text] can be selectively nucleated for even the mildest supercooling; 3) the water contact layer's resemblance to a face of ice is the key factor determining the polytype selectivity and nucleation temperature, independent of which polytype is promoted; and 4) substrate lattice match to ice is not indicative of the polytype obtained. Through this study, we have deepened understanding of the interplay of heterogeneous nucleation and ice I polytypism and suggest routes to [Formula: see text] More broadly, the substrate design methodology presented here combined with the insight gained can be used to understand and control polymorphism and stacking disorder in materials in general.

11.
Cancer Discov ; 2021 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-33707233

RESUMO

Many patients with advanced melanoma are resistant to immune checkpoint inhibition. In the ILLUMINATE-204 phase 1/2 trial, we assessed intratumoral tilsotolimod, an investigational Toll-like receptor 9 agonist, with systemic ipilimumab in patients with anti-PD-1-resistant advanced melanoma. In all patients, 48.4% experienced grade 3/4 treatment-emergent adverse events. The overall response rate at the recommended phase 2 dose of 8 mg was 22.4%, and an additional 49% of patients had stable disease. Responses in non-injected lesions and in patients expected to be resistant to ipilimumab monotherapy were observed. Rapid induction of a local interferon-alpha gene signature, dendritic cell maturation and enhanced markers of antigen presentation, and T-cell clonal expansion correlated with clinical response. A phase 3 clinical trial with this combination (NCT03445533) is ongoing.

12.
Free Radic Biol Med ; 167: 258-270, 2021 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-33731307

RESUMO

The present work examined the oxidation and crosslinking of the anti-bacterial enzyme lysozyme (Lyso), which is present in multiple biological fluids, and released from the cytoplasmic granules of macrophages and neutrophils at sites of infection and inflammation. It is therefore widely exposed to oxidants including peroxyl radicals (ROO•). We hypothesized that exposure to ROO• would generate specific modifications and inter- and intra-protein crosslinks via radical-radical reactions. Lyso was incubated with AAPH (2,2'-azobis(2-methylpropionamidine) dihydrochloride) as a ROO• source. Enzymatic activity was assessed, while oxidative modifications were detected and quantified using electrophoresis and liquid chromatography (UPLC) with fluorescence or mass detection (MS). Computational models of AAPH-Lyso interactions were developed. Exposure of Lyso to AAPH (10 and 100 mM for 3 h, and 20 mM for 1 h), at 37 °C, decreased enzymatic activity. 20 mM AAPH showed the highest efficiency of Lyso inactivation (1.78 mol of Lyso inactivated per ROO•). Conversion of Met to its sulfoxide, and to a lesser extent, Tyr oxidation to 3,4-dihydroxyphenylalanine and diTyr, were detected by UPLC-MS. Extensive transformation of Trp, involving short chain reactions, to kynurenine, oxindole, hydroxytryptophan, hydroperoxides or di-alcohols, and N-formyl-kynurenine was detected, with Trp62, Trp63 and Trp108 the most affected residues. Interactions of AAPH inside the negatively-charged catalytic pocket of Lyso, with Trp108, Asp52, and Glu35, suggest that Trp108 oxidation mediates, at least partly, Lyso inactivation. Crosslinks between Tyr20-Tyr23 (intra-molecular), and Trp62-Tyr23 (inter-molecular), were detected with both proximity (Tyr20-Tyr23), and chain flexibility (Trp62) appearing to favor the formation of covalent crosslinks.

13.
Nat Med ; 27(2): 301-309, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33558722

RESUMO

The association among pathological response, recurrence-free survival (RFS) and overall survival (OS) with neoadjuvant therapy in melanoma remains unclear. In this study, we pooled data from six clinical trials of anti-PD-1-based immunotherapy or BRAF/MEK targeted therapy. In total, 192 patients were included; 141 received immunotherapy (104, combination of ipilimumab and nivolumab; 37, anti-PD-1 monotherapy), and 51 received targeted therapy. A pathological complete response (pCR) occurred in 40% of patients: 47% with targeted therapy and 33% with immunotherapy (43% combination and 20% monotherapy). pCR correlated with improved RFS (pCR 2-year 89% versus no pCR 50%, P < 0.001) and OS (pCR 2-year OS 95% versus no pCR 83%, P = 0.027). In patients with pCR, near pCR or partial pathological response with immunotherapy, very few relapses were seen (2-year RFS 96%), and, at this writing, no patient has died from melanoma, whereas, even with pCR from targeted therapy, the 2-year RFS was only 79%, and OS was only 91%. Pathological response should be an early surrogate endpoint for clinical trials and a new benchmark for development and approval in melanoma.


Assuntos
Melanoma/tratamento farmacológico , Recidiva Local de Neoplasia/tratamento farmacológico , Proteínas Proto-Oncogênicas B-raf/genética , Neoplasias Cutâneas/tratamento farmacológico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Intervalo Livre de Doença , Feminino , Humanos , Imunoterapia/efeitos adversos , Ipilimumab/administração & dosagem , Ipilimumab/efeitos adversos , Masculino , Melanoma/genética , Melanoma/imunologia , Melanoma/patologia , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Terapia Neoadjuvante/efeitos adversos , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/imunologia , Recidiva Local de Neoplasia/patologia , Nivolumabe/administração & dosagem , Nivolumabe/efeitos adversos , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/genética , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/patologia , Adulto Jovem
14.
Free Radic Biol Med ; 166: 53-66, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33588048

RESUMO

Oxidation and inactivation of FtsZ is of interest due to the key role of this protein in bacterial cell division. In the present work, we studied peroxyl radical (from AAPH, 2,2'-azobis(2-methylpropionamidine)dihydrochloride) mediated oxidation of the highly stable FtsZ protein (MjFtsZ) from M. jannaschii, a thermophilic microorganism. MjFtsZ contains eleven Met, and single Tyr and Trp residues which would be expected to be susceptible to oxidation. We hypothesized that exposure of MjFtsZ to AAPH-derived radicals would induce Met oxidation, and cross-linking (via di-Tyr and di-Trp formation), with concomitant loss of its functional polymerization and depolymerization (GTPase) activities. Solutions containing MjFtsZ and AAPH (10 or 100 mM) were incubated at 37 °C for 3 h. Polymerization/depolymerization were assessed by light scattering, while changes in mass were analyzed by SDS-PAGE. Amino acid consumption was quantified by HPLC with fluorescence detection, or direct fluorescence (Trp). Oxidation products and modifications at individual Met residues were quantified by UPLC with mass detection. Oxidation inhibited polymerization-depolymerization activity, and yielded low levels of irreversible protein dimers. With 10 mM AAPH only Trp and Met were consumed giving di-alcohols, kynurenine and di-Trp (from Trp) and the sulfoxide (from Met). With 100 mM AAPH low levels of Tyr oxidation (but not di-Tyr formation) were also observed. Correlation with the functional analyses indicates that Met oxidation, and particularly Met164 is the key driver of MjFtsZ inactivation, probably as a result of the position of this residue at the protein-protein interface of longitudinal interactions and in close proximity to the GTP binding site.

15.
Free Radic Biol Med ; 166: 165-177, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33631301

RESUMO

Myeloperoxidase (MPO) is released by activated immune cells and forms the oxidants hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN) from the competing substrates chloride and thiocyanate. MPO and the overproduction of HOCl are strongly linked with vascular cell dysfunction and inflammation in atherosclerosis. HOCl is highly reactive and causes marked cell dysfunction and death, whereas data with HOSCN are conflicting, and highly dependent on the nature of the cell type. In this study we have examined the reactivity of HOCl and HOSCN with human coronary artery smooth muscle cells (HCASMC), given the key role of this cell type in maintaining vascular function. HOCl reacts rapidly with the cells, resulting in extensive cell death by both necrosis and apoptosis, and increased levels of intracellular calcium. In contrast, HOSCN reacts more slowly, with cell death occurring only after prolonged incubation, and in the absence of the accumulation of intracellular calcium. Exposure of HCASMC to HOCl also influences mitochondrial respiration, decreases glycolysis, lactate release, the production of ATP, cellular thiols and glutathione levels. These changes occurred to varying extents on exposure of the cells to HOSCN, where evidence was also obtained for the reversible modification of cellular thiols. HOCl also induced alterations in the mRNA expression of multiple inflammatory and phenotypic genes. Interestingly, the extent and nature of these changes was highly dependent on the specific cell donor used, with more marked effects observed in cells isolated from diseased compared to healthy vessels. Overall, these data provide new insight into pathways promoting vascular dysfunction during chronic inflammation, support the use of thiocyanate as a means to modulate MPO-induced cellular damage in atherosclerosis.

16.
Redox Biol ; 41: 101873, 2021 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-33550113

RESUMO

The production of hypochlorous acid (HOCl) by myeloperoxidase (MPO) plays a key role in immune defense, but also induces host tissue damage, particularly in chronic inflammatory pathologies, including atherosclerosis. This has sparked interest in the development of therapeutic approaches that decrease HOCl formation during chronic inflammation, including the use of alternative MPO substrates. Thiocyanate (SCN-) supplementation decreases HOCl production by favouring formation of hypothiocyanous acid (HOSCN), which is more selectively toxic to bacterial cells. Selenium-containing compounds are also attractive therapeutic agents as they react rapidly with HOCl and can be catalytically recycled. In this study, we examined the ability of SCN-, selenocyanate (SeCN-) and selenomethionine (SeMet) to modulate HOCl-induced damage to human coronary artery smooth muscle cells (HCASMC), which are critical to both normal vessel function and lesion formation in atherosclerosis. Addition of SCN- prevented HOCl-induced cell death, altered the pattern and extent of intracellular thiol oxidation, and decreased perturbations to calcium homeostasis and pro-inflammatory signaling. Protection was also observed with SeCN- and SeMet, though SeMet was less effective than SeCN- and SCN-. Amelioration of damage was detected with sub-stoichiometric ratios of the added compound to HOCl. The effects of SCN- are consistent with conversion of HOCl to HOSCN. Whilst SeCN- prevented HOCl-induced damage to a similar extent to SCN-, the resulting product hyposelenocyanous acid (HOSeCN), was more toxic to HCASMC than HOSCN. These results provide support for the use of SCN- and/or selenium analogues as scavengers, to decrease HOCl-induced cellular damage and HOCl production at inflammatory sites in atherosclerosis and other pathologies.

17.
Redox Biol ; 41: 101874, 2021 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-33601275

RESUMO

Cross-links formed within and between proteins are a major cause of protein dysfunction, and are postulated to drive the accumulation of protein aggregates in some human pathologies. Cross-links can be formed from multiple residues and can be reversible (usually sulfur-sulfur bonds) or irreversible (typically carbon-carbon or carbon-heteroatom bonds). Disulfides formed from oxidation of two Cys residues are widespread, with these formed both deliberately, via enzymatic reactions, or as a result of unintended oxidation reactions. We have recently demonstrated that new protein-glutathione mixed disulfides can be formed through oxidation of a protein disulfide to a thiosulfinate, and subsequent reaction of this species with glutathione. Here we investigate whether similar reactions occur between an oxidized protein disulfide, and a Cys residues on a second protein, to give novel protein cross-links. Singlet oxygen (1O2)-mediated oxidation of multiple proteins (α-lactalbumin, lysozyme, beta-2-microglobulin, C-reactive protein), and subsequent incubation with the Cys-containing protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH), generates inter-protein cross-links as detected by SDS-PAGE, immunoblotting and mass spectrometry (MS). The cross-link yield is dependent on the 1O2 concentration, the presence of the original protein disulfide bond, and the free Cys on GAPDH. MS with 18O-labeling has allowed identification of the residues involved in some cases (e.g. Cys25 from the Cys25-Cys80 disulfide in beta-2-microglobulin, with Cys149 or Cys244 of GAPDH). The formation of these cross-links results in a loss of GAPDH enzymatic activity. These data provide 'proof-of-concept' for a novel mechanism of protein cross-link formation which may help rationalize the accumulation of cross-linked proteins in multiple human pathologies.

18.
Lancet Oncol ; 22(3): 370-380, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33587894

RESUMO

BACKGROUND: Melanoma lacks validated blood-based biomarkers for monitoring and predicting treatment efficacy. Cell-free circulating tumour DNA (ctDNA) is a promising biomarker; however, various detection methods have been used, and, to date, no large studies have examined the association between serial changes in ctDNA and survival after BRAF, MEK, or BRAF plus MEK inhibitor therapy. We aimed to evaluate whether baseline ctDNA concentrations and kinetics could predict survival outcomes. METHODS: In this clinical validation study, we used analytically validated droplet digital PCR assays to measure BRAFV600-mutant ctDNA in pretreatment and on-treatment plasma samples from patients aged 18 years or older enrolled in two clinical trials. COMBI-d (NCT01584648) was a double-blind, randomised phase 3 study of dabrafenib plus trametinib versus dabrafenib plus placebo in previously untreated patients with BRAFV600 mutation-positive unresectable or metastatic melanoma. Patients had an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1. COMBI-MB (NCT02039947) was an open-label, non-randomised, phase 2 study evaluating dabrafenib plus trametinib in patients with BRAFV600 mutation-positive metastatic melanoma and brain metastases. Patients in cohort A of COMBI-MB had asymptomatic brain metastases, no previous local brain-directed therapy, and an ECOG performance status of 0 or 1. Biomarker analysis was a prespecified exploratory endpoint in both trials and performed in the intention-to-treat populations in COMBI-d and COMBI-MB. We investigated the association between mutant copy number (baseline or week 4 or zero conversion status) and efficacy endpoints (progression-free survival, overall survival, and best overall response). We used Cox models, Kaplan-Meier plots, and log-rank tests to explore the association of pretreatment ctDNA concentrations with progression-free survival and overall survival. The effect of additional prognostic variables such as lactate dehydrogenase was also investigated in addition to the mutant copy number. FINDINGS: In COMBI-d, pretreatment plasma samples were available from 345 (82%) of 423 patients and on-treatment (week 4) plasma samples were available from 224 (53%) of 423 patients. In cohort A of COMBI-MB, pretreatment and on-treatment samples were available from 38 (50%) of 76 patients with intracranial and extracranial metastatic melanoma. ctDNA was detected in pretreatment samples from 320 (93%) of 345 patients (COMBI-d) and 34 (89%) of 38 patients (COMBI-MB). When assessed as a continuous variable, elevated baseline BRAFV600 mutation-positive ctDNA concentration was associated with worse overall survival outcome (hazard ratio [HR] 1·13 [95% CI 1·09-1·18], p<0·0001 by univariate analysis), independent of treatment group and baseline lactate dehydrogenase concentrations (1·08 [1·03-1·13], p=0·0020), in COMBI-d. A ctDNA cutoff point of 64 copies per mL of plasma stratified patients enrolled in COMBI-d as high risk or low risk with respect to survival outcomes (HR 1·74 [95% CI 1·37-2·21], p<0·0001 for progression-free survival; 2·23 [1·73-2·87], p<0·0001 for overall survival) and was validated in the COMBI-MB cohort (3·20 [1·39-7·34], p=0·0047 for progression-free survival; 2·94 [1·18-7·32], p=0·016 for overall survival). In COMBI-d, undetectable ctDNA at week 4 was significantly associated with extended progression-free and overall survival, particularly in patients with elevated lactate dehydrogenase concentrations (HR 1·99 [95% CI 1·08-3·64], p=0·027 for progression-free survival; 2·38 [1·24-4·54], p=0·0089 for overall survival). INTERPRETATION: Pretreatment and on-treatment BRAFV600-mutant ctDNA measurements could serve as independent, predictive biomarkers of clinical outcome with targeted therapy. FUNDING: Novartis.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Encefálicas/secundário , DNA Tumoral Circulante/genética , Melanoma/patologia , Idoso , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , DNA Tumoral Circulante/análise , Método Duplo-Cego , Feminino , Seguimentos , Humanos , Imidazóis/administração & dosagem , Masculino , Melanoma/tratamento farmacológico , Melanoma/genética , Pessoa de Meia-Idade , Oximas/administração & dosagem , Prognóstico , Piridonas/administração & dosagem , Pirimidinonas/administração & dosagem , Taxa de Sobrevida
19.
J Gen Physiol ; 153(3)2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33533890

RESUMO

The dynein motor protein transports proteins away from the cell membrane along the microtubule network. Recently, we found the microtubule network was important for regulating the membrane abundance of voltage-gated Kv7.4 potassium channels in vascular smooth muscle. Here, we aimed to investigate the influence of dynein on the microtubule-dependent internalization of the Kv7.4 channel. Patch-clamp recordings from HEK293B cells showed Kv7.4 currents were increased after inhibiting dynein function with ciliobrevin D or by coexpressing p50/dynamitin, which specifically interferes with dynein motor function. Mutation of a dynein-binding site in the Kv7.4 C terminus increased the Kv7.4 current and prevented p50 interference. Structured illumination microscopy, proximity ligation assays, and coimmunoprecipitation showed colocalization of Kv7.4 and dynein in mesenteric artery myocytes. Ciliobrevin D enhanced mesenteric artery relaxation to activators of Kv7.2-Kv7.5 channels and increased membrane abundance of Kv7.4 protein in isolated smooth muscle cells and HEK293B cells. Ciliobrevin D failed to enhance the negligible S-1-mediated relaxations after morpholino-mediated knockdown of Kv7.4. Mass spectrometry revealed an interaction of dynein with caveolin-1, confirmed using proximity ligation and coimmunoprecipitation assays, which also provided evidence for interaction of caveolin-1 with Kv7.4, confirming that Kv7.4 channels are localized to caveolae in mesenteric artery myocytes. Lastly, cholesterol depletion reduced the interaction of Kv7.4 with caveolin-1 and dynein while increasing the overall membrane expression of Kv7.4, although it attenuated the Kv7.4 current in oocytes and interfered with the action of ciliobrevin D and channel activators in arterial segments. Overall, this study shows that dynein can traffic Kv7.4 channels in vascular smooth muscle in a mechanism dependent on cholesterol-rich caveolae.

20.
J Biol Chem ; : 100360, 2021 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-33539924

RESUMO

Fibronectin (FN) is an abundant glycoprotein found in plasma and the extracellular matrix (ECM). It is present at high concentrations at sites of tissue damage, where is it exposed to oxidants generated by activated leukocytes, including peroxynitrous acid (ONOOH) formed from nitric oxide (from inducible nitric oxide synthase) and superoxide radicals (from NADPH oxidases and other sources). ONOOH reacts rapidly with the abundant tyrosine and tryptophan residues in ECM proteins, resulting in the formation of 3-nitroTyr, di-tyrosine, and 6-nitrotryptophan (6-nitroTrp). We have shown previously that human plasma fibronectin is readily modified by ONOOH, but the extent and location of modifications, and the role of fibronectin structure (compact versus extended) in determining these factors is poorly understood. Here we provide a detailed LC-MS analysis of ONOOH-induced fibronectin modifications, including the extent of their formation and the sites of intra- and inter-molecular cross-links, including Tyr-Tyr, Trp-Trp and Tyr-Trp linkages. The localization of these cross-links to specific domains provides novel data on the interactions between different modules in the compact conformation of plasma FN, and allows us to propose a model of its unknown quaternary structure. Interestingly, the pattern of modifications is significantly different to that generated by another inflammatory oxidant, HOCl, in both extent and sites. The characterization and quantification of these modifications offers the possibility of the use of these materials as specific biomarkers of extracellular matrix modification and turnover in the many pathologies associated with inflammation-associated fibrosis.

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