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Introduction: Prescription and most over-the-counter medicines are required to have child-resistant packaging and/or labeled with instructions "Keep out of reach of children." Although medication organizers are not required to have such design features or instructions, these could help prevent unsupervised ingestions by children. Commonly purchased medication organizers were evaluated for child-resistant design features and instructions for safe use to prevent unsupervised ingestions. Methods: The 29 best-selling medication organizers on Amazon.com were identified, and product identifiers, design characteristics, and safety characteristics were recorded using a standardized instrument. Results: Of the 29 medication organizers, none claimed to be child resistant. Only 31% provided a specific warning that the organizer was not child resistant on the packaging; only 41% communicated "Keep out of reach of children." Most organizers (59%) provided neither a warning that the organizer was not child resistant nor instructions to store out of reach of children. The majority of organizers (79%) shared the following characteristics: plastic construction, rectangular shape, nonelectronic flip-top opening mechanisms, and 7-day usage. Conclusions: Opportunities exist for manufacturers of medication organizers to improve child-resistant product design, provide information to help prevent unsupervised ingestions (directions to keep the device out of the reach of children), and help to reduce unsupervised ingestions.
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The mechanistic target of rapamycin (mTOR) signaling is influenced by multiple regulatory proteins and post-translational modifications; however, underlying mechanisms remain unclear. Here, we report a novel role of small ubiquitin-like modifier (SUMO) in mTOR complex assembly and activity. By investigating the SUMOylation status of core mTOR components, we observed that the regulatory subunit, GßL (G protein ß-subunit-like protein, also known as mLST8), is modified by SUMO1, 2, and 3 isoforms. Using mutagenesis and mass spectrometry, we identified that GßL is SUMOylated at lysine sites K86, K215, K245, K261, and K305. We found that SUMO depletion reduces mTOR-Raptor (regulatory protein associated with mTOR) and mTOR-Rictor (rapamycin-insensitive companion of mTOR) complex formation and diminishes nutrient-induced mTOR signaling. Reconstitution with WT GßL but not SUMOylation-defective KR mutant GßL promotes mTOR signaling in GßL-depleted cells. Taken together, we report for the very first time that SUMO modifies GßL, influences the assembly of mTOR protein complexes, and regulates mTOR activity.
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Transdução de Sinais , Sumoilação , Serina-Treonina Quinases TOR , Humanos , Serina-Treonina Quinases TOR/metabolismo , Serina-Treonina Quinases TOR/genética , Células HEK293 , Proteína SUMO-1/metabolismo , Proteína SUMO-1/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Homólogo LST8 da Proteína Associada a mTOR/metabolismo , Homólogo LST8 da Proteína Associada a mTOR/genética , Ubiquitinas/metabolismo , Ubiquitinas/genética , Lisina/metabolismoRESUMO
AIM: The aim of this study is to explore experiences and perspectives of nurses and providers (e.g., physicians, medical directors, fellows, and nurse practitioners) on reducing preventable hospitalizations of nursing home (NH) residents in relation to interprofessional relationship and hospitalization decision-making process. BACKGROUND: Preventable NH resident hospitalization continues to be a pressing public health issue. Studies show that improved interprofessional relationship may help reduce hospitalization, yet research on communication processes and interactions among different NH staff remains limited. METHODS: This is a qualitative descriptive study. Two focus groups were held with fourteen nurses and thirteen in-depth, qualitative interviews were conducted with providers from two Chicagoland NHs. Focus group sessions and interviews were transcribed, coded, and analyzed for common themes based on qualitative description method. RESULTS: All study participants agreed that providers have the ultimate responsibility for hospitalization decisions. However, nurses believed they could influence those decisions, depending on provider characteristics, trust, and resident conditions. Nurses and providers differed in the way they experienced and conveyed emotions, and differed in key elements affecting hospitalization decisions such as structural or environmental factors (e.g., lacking staff and equipment at the facility, poor communication between the NH and hospitals) and interpersonal factors (e.g., characteristics of effective nurses or providers and the effective interactions between them). CONCLUSIONS: Interpersonal factors, including perceived competence, respect, and trust, may influence NH hospitalization decisions and be targeted for reducing preventable hospitalizations of residents.
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Recursos Humanos de Enfermagem , Médicos , Humanos , Hospitalização , Casas de Saúde , Hospitais , Pesquisa QualitativaRESUMO
Mutations in genes coding for proteasome subunits and/or proteasome assembly helpers typically cause recurring autoinflammation referred to as chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperatures (CANDLE) or proteasome-associated autoinflammatory syndrome (PRAAS). Patients with CANDLE/PRAAS present with mostly chronically elevated type I interferon scores that emerge as a consequence of increased proteotoxic stress by mechanisms that are not fully understood. Here, we report on five unrelated patients with CANDLE/PRAAS carrying novel inherited proteasome missense and/or nonsense variants. Four patients were compound heterozygous for novel pathogenic variants in the known CANDLE/PRAAS associated genes, PSMB8 and PSMB10, whereas one patient showed additive loss-of-function mutations in PSMB8. Variants in two previously not associated proteasome genes, PSMA5 and PSMC5, were found in a patient who also carried the PSMB8 founder mutation, p.T75M. All newly identified mutations substantially impact the steady-state expression of the affected proteasome subunits and/or their incorporation into mature 26S proteasomes. Our observations expand the spectrum of PRAAS-associated genetic variants and improve a molecular diagnosis and genetic counseling of patients with sterile autoinflammation.
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Dermatite , Complexo de Endopeptidases do Proteassoma , Humanos , Complexo de Endopeptidases do Proteassoma/genética , Síndrome , CitoplasmaRESUMO
OBJECTIVE: To characterise the extent of unnecessary care in general surgery inpatients using a triple bottom line approach. DESIGN: Patients with uncomplicated acute surgical conditions were retrospectively evaluated for unnecessary bloodwork according to the triple bottom line, quantifying the impacts on patients, healthcare costs and greenhouse gas emissions. The carbon footprint of common laboratory investigations was estimated using PAS2050 methodology, including emissions generated from the production, transport, processing and disposal of consumable goods and reagents. SETTING: Single-centre tertiary care hospital. PARTICIPANTS: Patients admitted with acute uncomplicated appendicitis, cholecystitis, choledocholithiasis, gallstone pancreatitis and adhesive small bowel obstruction were included in the study. 304 patients met inclusion criteria and 83 were randomly selected for in-depth chart review. MAIN OUTCOME MEASURES: In each patient population, the extent of over-investigation was determined by comparing ordered laboratory investigations against previously developed consensus recommendations. The quantity of unnecessary bloodwork was measured by number of phlebotomies, tests and blood volume in addition to healthcare costs and greenhouse gas emissions. RESULTS: 76% (63/83) of evaluated patients underwent unnecessary bloodwork resulting in a mean of 1.84 phlebotomies, 4.4 blood vials, 16.5 tests and 18 mL of blood loss per patient. The hospital and environmental cost of these unnecessary activities was $C5235 and 61 kg CO2e (974 g CO2e per person), respectively. The carbon footprint of a common set of investigations (complete blood count, differential, creatinine, urea, sodium, potassium) was 332 g CO2e. Adding a liver panel (liver enzymes, bilirubin, albumin, international normalised ratio/partial thromboplastin time) resulted in an additional 462 g CO2e. CONCLUSIONS: We found considerable overuse of laboratory investigations among general surgery patients admitted with uncomplicated acute surgical conditions resulting in unnecessary burden to patients, hospitals and the environment. This study identifies an opportunity for resource stewardship and exemplifies a comprehensive approach to quality improvement.
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Gases de Efeito Estufa , Humanos , Estudos Retrospectivos , Pegada de Carbono , Hospitalização , HospitaisRESUMO
People with disabilities represent the largest minority group in the United States and a priority population for health services research. Despite federal civil rights law, people with disabilities face inaccessible health care environments that fail to accommodate their disability. We present Michigan Medicine's Disability and Accommodations Tab. This patient-facing questionnaire and shared data field in the electronic health record enables the collection and reporting of patient disability-related accommodations. The Disability Tab seeks to address provider- and clinic staff-reported barriers to providing accommodations and fosters an opportunity to redesign health care to meet the needs of people with disabilities.
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Molecularly targeted therapeutics have revolutionized the treatment of BRAFV600E -driven malignant melanoma, but the rapid development of resistance to BRAF kinase inhibitors (BRAFi) presents a significant obstacle. The use of clinical antimalarials for the investigational treatment of malignant melanoma has shown only moderate promise, attributed mostly to inhibition of lysosomal-autophagic adaptations of cancer cells, but identification of specific antimalarials displaying single-agent antimelanoma activity has remained elusive. Here, we have screened a focused library of clinically used artemisinin-combination therapeutic (ACT) antimalarials for the apoptotic elimination of cultured malignant melanoma cell lines, also examining feasibility of overcoming BRAFi-resistance comparing isogenic melanoma cells that differ only by NRAS mutational status (BRAFi-sensitive A375-BRAFV600E /NRASQ61 vs. BRAFi-resistant A375-BRAFV600E /NRASQ61K ). Among ACT antimalarials tested, mefloquine (MQ) was the only apoptogenic agent causing melanoma cell death at low micromolar concentrations. Comparative gene expression-array analysis (A375-BRAFV600E /NRASQ61 vs. A375-BRAFV600E /NRASQ61K ) revealed that MQ is a dual inducer of endoplasmic reticulum (ER) and redox stress responses that precede MQ-induced loss of viability. ER-trackerTM DPX fluorescence imaging and electron microscopy indicated ER swelling, accompanied by rapid induction of ER stress signaling (phospho-eIF2α, XBP-1s, ATF4). Fluo-4 AM-fluorescence indicated the occurrence of cytosolic calcium overload observable within seconds of MQ exposure. In a bioluminescent murine model employing intracranial injection of A375-Luc2 (BRAFV600E /NRASQ61K ) cells, an oral MQ regimen efficiently antagonized brain tumor growth. Taken together, these data suggest that the clinical antimalarial MQ may be a valid candidate for drug repurposing aiming at chemotherapeutic elimination of malignant melanoma cells, even if metastasized to the brain and BRAFi-resistant.
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Antimaláricos , Neoplasias Encefálicas , Melanoma , Animais , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Apoptose , Neoplasias Encefálicas/tratamento farmacológico , Linhagem Celular Tumoral , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/genética , GTP Fosfo-Hidrolases/genética , Humanos , Mefloquina/farmacologia , Mefloquina/uso terapêutico , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/patologia , Proteínas de Membrana/genética , Camundongos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf , Neoplasias Cutâneas , Melanoma Maligno CutâneoRESUMO
Background and Aims: Fecal calprotectin (FC) is a noninvasive biomarker used in inflammatory bowel disease (IBD) management and risk stratification of nonspecific gastrointestinal symptoms. Leukocyte esterase is an inexpensive and widely available point-of-care inflammatory marker present on urinalysis test strips. We aim to assess the diagnostic accuracy of fecal leukocyte esterase (FLE) relative to FC and endoscopy and demonstrate its use as an alternative biomarker for IBD. Methods: In this prospective cohort study, 70 patients who had FC ordered as part of standard clinical care also received FLE testing. FLE levels were compared with various FC cutoff values and endoscopy and pathology findings as the gold standard. Results: As the FC cutoff increased from 50 to 500 µg/g, FLE sensitivity increased from 67% to 95% while the specificity decreased from 86% to 76%. The area under the receiver operating characteristic (AUROC) curve increased from 0.79 to 0.90. An FLE of ≥1+ had the best test characteristics. Among patients who underwent endoscopic evaluation, FLE demonstrated an identical sensitivity (75%) and specificity (86%) to FC in predicting endoscopic inflammation. AUROC was 0.80 for FLE and 0.85 for FC with an optimal cutoff of ≥2+ and 301 µg/g, respectively. When used to distinguish between patients with active IBD and no/inactive IBD, FLE had a sensitivity of 84% and specificity of 90%, comparable with the 84% and 83%, respectively, of FC. AUROC was 0.88 for FLE and 0.91 for FC with an optimal cutoff of ≥2+ and 145 µg/g, respectively. Conclusion: FLE demonstrates adequate correlation and comparable accuracy with FC in predicting endoscopic inflammation and distinguishing between patients with active vs inactive IBD.
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The human GID (hGID) complex is a conserved E3 ubiquitin ligase regulating diverse biological processes, including glucose metabolism and cell cycle progression. However, the biochemical function and substrate recognition of the multi-subunit complex remain poorly understood. Using biochemical assays, cross-linking mass spectrometry, and cryo-electron microscopy, we show that hGID engages two distinct modules for substrate recruitment, dependent on either WDR26 or GID4. WDR26 and RanBP9 cooperate to ubiquitinate HBP1 in vitro, while GID4 is dispensable for this reaction. In contrast, GID4 functions as an adaptor for the substrate ZMYND19, which surprisingly lacks a Pro/N-end degron. GID4 substrate binding and ligase activity is regulated by ARMC8α, while the shorter ARMC8ß isoform assembles into a stable hGID complex that is unable to recruit GID4. Cryo-EM reconstructions of these hGID complexes reveal the localization of WDR26 within a ring-like, tetrameric architecture and suggest that GID4 and WDR26/Gid7 utilize different, non-overlapping binding sites. Together, these data advance our mechanistic understanding of how the hGID complex recruits cognate substrates and provides insights into the regulation of its E3 ligase activity.
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Proteínas de Grupo de Alta Mobilidade , Ubiquitina-Proteína Ligases , Sítios de Ligação , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Microscopia Crioeletrônica , Proteínas de Grupo de Alta Mobilidade/metabolismo , Humanos , Proteínas Repressoras/metabolismo , Especificidade por Substrato , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
PURPOSE: To provide an overview of compounding under sections 503A and 503B of the Federal Food, Drug, and Cosmetic Act, and to describe the pharmacist's role within the US Food and Drug Administration's (FDA's) Compounding Incidents Program, whose efforts are aimed at protecting the public against poor-quality compounded drugs through surveillance, review and response to adverse events and complaints. SUMMARY: Compounded drugs may serve an important medical need for patients who cannot be treated with medications approved by FDA; however, compounded drugs are not approved by FDA and are not subject to premarket review for safety, efficacy, or manufacturing quality; thus, they may pose safety risks to patients. Prompt reporting of adverse events or complaints related to compounding is important in identifying these risks and implementing safeguards to protect the public. FDA's Compounding Incidents Program consists of a team of pharmacists dedicated to the surveillance and review of adverse events and complaints and follow-up actions related to safety risks associated with compounded drugs. Pharmacists are a vital component of FDA's Compounding Incidents Program, utilizing their clinical skill set and regulatory knowledge to review and act on safety issues that affect public health. CONCLUSION: As FDA continues to expand the Compounding Incidents Program and its efforts to protect the public against poor-quality compounded drugs, we encourage the continued submission of adverse event reports by healthcare professionals and consumers to FDA's MedWatch reporting system in addition to adverse event reporting compliance by outsourcing facilities.
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Preparações Farmacêuticas , Farmacêuticos , Composição de Medicamentos , Humanos , Estados Unidos , United States Food and Drug AdministrationRESUMO
Antibody-dependent cellular cytotoxicity (ADCC) is the primary mechanism of actions for several marketed therapeutic antibodies (mAbs) and for many more in clinical trials. The ADCC efficacy is highly dependent on the ability of therapeutic mAbs to recruit effector cells such as natural killer cells, which induce the apoptosis of targeted cells. The recruitment of effector cells by mAbs is negatively affected by fucose modification of N-Glycans on the Fc; thus, utilization of afucosylated mAbs has been a trend for enhanced ADCC therapeutics. Most of afucosylated mAbs in clinical or commercial manufacturing were produced from Fut8-/- Chinese hamster ovary cells (CHO) host cells, generally generating low yields compared to wildtype CHO host. This study details the generation and characterization of two engineered CHOZN® cell lines, in which the enzyme involved in guanosine diphosphate (GDP)-fucose synthesis, GDP mannose-4,6-dehydratase (Gmds) and GDP-L-fucose synthase (FX), was knocked out. The top host cell lines for each of the knockouts, FX-/- and Gmds-/-, were selected based on growth robustness, bulk MSX selection tolerance, production titer, fucosylation level, and cell stability. We tested the production of two proprietary IgG1 mAbs in the engineered host cells, and found that the titers were comparable to CHOZN® cells. The mAbs generated from either KO cell line exhibited loss of fucose modification, leading to significantly boosted FcγRIIIa binding and ADCC effects. Our data demonstrated that both FX-/- and Gmds-/- host cells could replace Fut8-/- CHO cells for clinical manufacturing of antibody therapeutics.
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Anticorpos Monoclonais/biossíntese , Carboidratos Epimerases/antagonistas & inibidores , Fucose/metabolismo , Guanosina Difosfato/metabolismo , Hidroliases/antagonistas & inibidores , Cetona Oxirredutases/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Citotoxicidade Celular Dependente de Anticorpos , Sequência de Bases , Células CHO , Sistemas CRISPR-Cas , Carboidratos Epimerases/genética , Carboidratos Epimerases/metabolismo , Cricetinae , Cricetulus , Glicosilação , Humanos , Hidroliases/genética , Hidroliases/metabolismo , Imunoglobulina G/imunologia , Cetona Oxirredutases/genética , Cetona Oxirredutases/metabolismo , Receptores de IgG/metabolismoRESUMO
UV-chromophores contained in human skin may act as endogenous sensitizers of photooxidative stress and can be employed therapeutically for the photodynamic elimination of malignant cells. Here, we report that 6-formylindolo[3,2-b]carbazole (FICZ), a tryptophan-derived photoproduct and endogenous aryl hydrocarbon receptor agonist, displays activity as a nanomolar sensitizer of photooxidative stress, causing the photodynamic elimination of human melanoma and nonmelanoma skin cancer cells in vitro and in vivo. FICZ is an efficient UVA/Visible photosensitizer having absorbance maximum at 390 nm (ε = 9180 L mol-1 cm-1 ), and fluorescence and singlet oxygen quantum yields of 0.15 and 0.5, respectively, in methanol. In a panel of cultured human squamous cell carcinoma and melanoma skin cancer cells (SCC-25, HaCaT-ras II-4, A375, G361, LOX), photodynamic induction of cell death was elicited by the combined action of solar simulated UVA (6.6 J cm-2 ) and FICZ (≥10 nm), preceded by the induction of oxidative stress as substantiated by MitoSOX Red fluorescence microscopy, comet detection of Fpg-sensitive oxidative genomic lesions and upregulated stress response gene expression (HMOX1, HSPA1A, HSPA6). In SKH1 "high-risk" mouse skin, an experimental FICZ/UVA photodynamic treatment regimen blocked the progression of UV-induced tumorigenesis suggesting feasibility of harnessing FICZ for the photooxidative elimination of malignant cells in vivo.
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Carcinoma de Células Escamosas/tratamento farmacológico , Melanoma/tratamento farmacológico , Fotoquimioterapia , Fármacos Fotossensibilizantes/farmacologia , Neoplasias Cutâneas/tratamento farmacológico , Triptofano/análogos & derivados , Animais , Carbazóis , Morte Celular , Linhagem Celular Tumoral , Epiderme/efeitos dos fármacos , Epiderme/efeitos da radiação , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/efeitos da radiação , Camundongos , Mitocôndrias , Estresse Oxidativo , Fármacos Fotossensibilizantes/química , Terapia UltravioletaRESUMO
OBJECTIVE: High comorbidity among psychiatric disorders suggests that they may share underlying neurobiological deficits. Abnormalities in cortical thickness and volume have been demonstrated in clinical samples of adults, but less is known when these structural differences emerge in youths. The purpose of this study was to examine the association between dimensions of psychopathology and brain structure. METHODS: The authors studied 1,394 youths who underwent brain imaging as part of the Philadelphia Neurodevelopmental Cohort. Dimensions of psychopathology were constructed using a bifactor model of symptoms. Cortical thickness and volume were quantified using high-resolution 3-T MRI. Structural covariance networks were derived using nonnegative matrix factorization and analyzed using generalized additive models with penalized splines to capture both linear and nonlinear age-related effects. RESULTS: Fear symptoms were associated with reduced cortical thickness in most networks, and overall psychopathology was associated with globally reduced gray matter volume across all networks. Structural covariance networks predicted psychopathology symptoms above and beyond demographic characteristics and cognitive performance. CONCLUSIONS: The results suggest a dissociable relationship whereby fear is most strongly linked to reduced cortical thickness and overall psychopathology is most strongly linked to global reductions in gray matter volume. Such results have implications for understanding how abnormalities of brain development may be associated with divergent dimensions of psychopathology.
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Córtex Cerebral/patologia , Substância Cinzenta/patologia , Transtornos Mentais/patologia , Adolescente , Atrofia/patologia , Criança , Cognição , Medo , Feminino , Humanos , Imageamento por Ressonância Magnética , Masculino , Transtornos Mentais/psicologia , Vias Neurais/patologia , Testes Neuropsicológicos , PsicopatologiaRESUMO
Redox-directed pharmacophores have shown potential for the apoptotic elimination of cancer cells through chemotherapeutic induction of oxidative stress. Phenazine methosulfate (PMS), a N-alkylphenazinium cation-based redox cycler, is used widely as an electron transfer reactant coupling NAD(P)H generation to the reduction of tetrazolium salts in biochemical cell viability assays. Here, we have explored feasibility of repurposing the redox cycler PMS as a superoxide generating chemotherapeutic for the pro-oxidant induction of cancer cell apoptosis. In a panel of malignant human melanoma cells (A375, G361, LOX), low micromolar concentrations of PMS (1-10 µM, 24 h) displayed pronounced apoptogenicity as detected by annexin V-ITC/propidium iodide flow cytometry, and PMS-induced cell death was suppressed by antioxidant (NAC) or pan-caspase inhibitor (zVAD-fmk) cotreatment. Gene expression array analysis in A375 melanoma cells (PMS, 10 µM; 6 h) revealed transcriptional upregulation of heat shock (HSPA6, HSPA1A), oxidative (HMOX1) and genotoxic (EGR1, GADD45A) stress responses, confirmed by immunoblot detection demonstrating upregulation of redox regulators (NRF2, HO-1, HSP70) and modulation of pro- (BAX, PUMA) and anti-apoptotic factors (Bcl-2, Mcl-1). PMS-induced oxidative stress and glutathione depletion preceded induction of apoptotic cell death. Furthermore, the mitochondrial origin of PMS-induced superoxide production was substantiated by MitoSOX-Red live cell fluorescence imaging, and PMS-induced mitochondriotoxicity (as evidenced by diminished transmembrane potential and oxygen consumption rate) was observable at early time points. After demonstrating NADPH-driven (SOD-suppressible) superoxide radical anion generation by PMS employing a chemical NBT reduction assay, PMS-induction of oxidative genotoxic stress was substantiated by quantitative Comet analysis that confirmed the introduction of formamido-pyrimidine DNA glycosylase (Fpg)-sensitive oxidative DNA lesions in A375 melanoma cells. Taken together, these data suggest feasibility of repurposing the biochemical reactant PMS as an experimental pro-oxidant targeting mitochondrial integrity and redox homeostasis for the apoptotic elimination of malignant melanoma cells.
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UVA-driven photooxidative stress in human skin may originate from excitation of specific endogenous chromophores acting as photosensitizers. Previously, we have demonstrated that 3-hydroxypyridine-derived chromophores including B6 -vitamers (pyridoxine, pyridoxamine and pyridoxal) are endogenous photosensitizers that enhance UVA-induced photooxidative stress in human skin cells. Here, we report that the B6 -vitamer pyridoxal is a sensitizer of genotoxic stress in human adult primary keratinocytes (HEKa) and reconstructed epidermis. Comparative array analysis indicated that exposure to the combined action of pyridoxal and UVA caused upregulation of heat shock (HSPA6, HSPA1A, HSPA1L, HSPA2), redox (GSTM3, EGR1, MT2A, HMOX1, SOD1) and genotoxic (GADD45A, DDIT3, CDKN1A) stress response gene expression. Together with potentiation of UVA-induced photooxidative stress and glutathione depletion, induction of HEKa cell death occurred only in response to the combined action of pyridoxal and UVA. In addition to activational phosphorylation indicative of genotoxic stress [p53 (Ser15) and γ-H2AX (Ser139)], comet analysis indicated the formation of Fpg-sensitive oxidative DNA lesions, observable only after combined exposure to pyridoxal and UVA. In human reconstructed epidermis, pyridoxal preincubation followed by UVA exposure caused genomic oxidative base damage, procaspase 3 cleavage and TUNEL positivity, consistent with UVA-driven photooxidative damage that may be relevant to human skin exposed to high concentrations of B6 -vitamers.
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Dano ao DNA , Epiderme/efeitos dos fármacos , Epiderme/efeitos da radiação , Queratinócitos/efeitos dos fármacos , Queratinócitos/efeitos da radiação , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/efeitos da radiação , Piridoxal/farmacologia , Raios Ultravioleta/efeitos adversos , Adulto , Células Cultivadas , Epiderme/metabolismo , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/efeitos da radiação , Humanos , Queratinócitos/metabolismoRESUMO
Exposure to solar ultraviolet (UV) radiation is a causative factor in skin photodamage and carcinogenesis, and an urgent need exists for improved molecular photoprotective strategies different from (or synergistic with) photon absorption. Recent studies suggest a photoprotective role of cutaneous gene expression orchestrated by the transcription factor NRF2 (nuclear factor-E2-related factor 2). Here we have explored the molecular mechanism underlying carotenoid-based systemic skin photoprotection in SKH-1 mice and provide genetic evidence that photoprotection achieved by the FDA-approved apocarotenoid and food additive bixin depends on NRF2 activation. Bixin activates NRF2 through the critical Cys-151 sensor residue in KEAP1, orchestrating a broad cytoprotective response in cultured human keratinocytes as revealed by antioxidant gene expression array analysis. Following dose optimization studies for cutaneous NRF2 activation by systemic administration of bixin, feasibility of bixin-based suppression of acute cutaneous photodamage from solar UV exposure was investigated in Nrf2(+/+) versus Nrf2(-/-) SKH-1 mice. Systemic administration of bixin suppressed skin photodamage, attenuating epidermal oxidative DNA damage and inflammatory responses in Nrf2(+/+) but not in Nrf2(-/-) mice, confirming the NRF2-dependence of bixin-based cytoprotection. Taken together, these data demonstrate feasibility of achieving NRF2-dependent cutaneous photoprotection by systemic administration of the apocarotenoid bixin, a natural food additive consumed worldwide.
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Carotenoides/farmacologia , Queratinócitos/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/metabolismo , Pele/efeitos dos fármacos , Animais , Sobrevivência Celular/efeitos dos fármacos , Citoproteção/fisiologia , Citometria de Fluxo , Humanos , Immunoblotting , Marcação In Situ das Extremidades Cortadas , Queratinócitos/efeitos da radiação , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estresse Oxidativo/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Pele/efeitos da radiação , Luz Solar/efeitos adversos , Transfecção , Raios Ultravioleta/efeitos adversosRESUMO
The progressive nature of colorectal cancer and poor prognosis associated with the metastatic phase of the disease create an urgent need for the development of more efficacious strategies targeting colorectal carcinogenesis. Cumulative evidence suggests that the redox-sensitive transcription factor Nrf2 (nuclear factor-E2-related factor 2), a master regulator of the cellular antioxidant defence, represents a promising molecular target for colorectal cancer chemoprevention. Recently, we have identified cinnamon, the ground bark of Cinnamomum aromaticum (cassia cinnamon) and Cinnamomum verum (Ceylon cinnamon), as a rich dietary source of the Nrf2 inducer cinnamaldehyde (CA) eliciting the Nrf2-regulated antioxidant response in human epithelial colon cells, conferring cytoprotection against electrophilic and genotoxic insult. Here, we have explored the molecular mechanism underlying CA-induced Nrf2 activation in colorectal epithelial cells and have examined the chemopreventive potential of CA in a murine colorectal cancer model comparing Nrf2(+/+) with Nrf2(-/-) mice. In HCT116 cells, CA caused a Keap1-C151-dependent increase in Nrf2 protein half-life via blockage of ubiquitination with upregulation of cytoprotective Nrf2 target genes and elevation of cellular glutathione. After optimizing colorectal Nrf2 activation and target gene expression by dietary CA-supplementation regimens, we demonstrated that CA suppresses AOM/DSS-induced inflammatory colon carcinogenesis with modulation of molecular markers of colorectal carcinogenesis. Dietary suppression of colorectal cancer using CA supplementation was achieved in Nrf2(+/+) but not in Nrf2(-/-) mice confirming the Nrf2 dependence of CA-induced chemopreventive effects. Taken together, our data suggest feasibility of colorectal cancer suppression by dietary CA, an FDA-approved food additive derived from the third most consumed spice in the world.
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Acroleína/análogos & derivados , Anticarcinógenos/farmacologia , Azoximetano , Carcinogênese/efeitos dos fármacos , Neoplasias do Colo/prevenção & controle , Sulfato de Dextrana , Fator 2 Relacionado a NF-E2/fisiologia , Acroleína/farmacologia , Acroleína/uso terapêutico , Animais , Carcinogênese/genética , Quimioprevenção/métodos , Cinnamomum zeylanicum/química , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Citoproteção/efeitos dos fármacos , Citoproteção/genética , Dieta , Células HCT116 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 2 Relacionado a NF-E2/genéticaRESUMO
Endogenous UVA chromophores may act as sensitizers of oxidative stress underlying cutaneous photoaging and photocarcinogenesis, but the molecular identity of non-DNA key chromophores displaying UVA-driven photodyamic activity in human skin remains largely undefined. Here we report that 6-formylindolo[3,2-b]carbazole (FICZ), a tryptophan photoproduct and endogenous high-affinity aryl hydrocarbon receptor (AhR) agonist, acts as a nanomolar photosensitizer potentiating UVA-induced oxidative stress irrespective of AhR ligand activity. In human HaCaT and primary epidermal keratinocytes, photodynamic induction of apoptosis was elicited by the combined action of solar-simulated UVA and FICZ, whereas exposure to the isolated action of UVA or FICZ did not impair viability. In a human epidermal tissue reconstruct, FICZ/UVA cotreatment caused pronounced phototoxicity inducing keratinocyte cell death, and FICZ photodynamic activity was also substantiated in a murine skin exposure model. Array analysis revealed pronounced potentiation of cellular heat shock, endoplasmic reticulum stress, and oxidative stress response gene expression observed only upon FICZ/UVA cotreatment. FICZ photosensitization caused intracellular oxidative stress, and comet analysis revealed introduction of formamidopyrimidine-DNA glycosylase (Fpg)-sensitive oxidative DNA lesions suppressible by antioxidant cotreatment. Taken together, our data demonstrate that the endogenous AhR ligand FICZ displays nanomolar photodynamic activity representing a molecular mechanism of UVA-induced photooxidative stress potentially operative in human skin.
Assuntos
Carbazóis/química , Queratinócitos/efeitos dos fármacos , Queratinócitos/efeitos da radiação , Fármacos Fotossensibilizantes/química , Pele/efeitos dos fármacos , Pele/efeitos da radiação , Triptofano/química , Animais , Antioxidantes/química , Linhagem Celular , DNA/química , DNA Glicosilases/metabolismo , Epiderme/efeitos dos fármacos , Epiderme/metabolismo , Epiderme/efeitos da radiação , Feminino , Regulação da Expressão Gênica , Humanos , Imuno-Histoquímica , Ligantes , Camundongos , Estresse Oxidativo , Fotoquimioterapia/métodos , Reação em Cadeia da Polimerase , Receptores de Hidrocarboneto Arílico/químicaRESUMO
Pharmacological induction of proteotoxic stress is rapidly emerging as a promising strategy for cancer cell-directed chemotherapeutic intervention. Here, we describe the identification of a novel drug-like heat shock response inducer for the therapeutic induction of proteotoxic stress targeting malignant human melanoma cells. Screening a focused library of compounds containing redox-directed electrophilic pharmacophores employing the Stress & Toxicity PathwayFinder(TM) PCR Array technology as a discovery tool, a drug-like triphenylmethane-derivative (aurin; 4-[bis(p-hydroxyphenyl)methylene]-2,5-cyclohexadien-1-one) was identified as an experimental cell stress modulator that causes (i) heat shock factor transcriptional activation, (ii) up-regulation of heat shock response gene expression (HSPA6, HSPA1A, DNAJB4, HMOX1), (iii) early unfolded protein response signaling (phospho-PERK, phospho-eIF2α, CHOP (CCAAT/enhancer-binding protein homologous protein)), (iv) proteasome impairment with increased protein-ubiquitination, and (v) oxidative stress with glutathione depletion. Fluorescence polarization-based experiments revealed that aurin displays activity as a geldanamycin-competitive Hsp90α-antagonist, a finding further substantiated by molecular docking and ATPase inhibition analysis. Aurin exposure caused caspase-dependent cell death in a panel of human malignant melanoma cells (A375, G361, LOX-IMVI) but not in non-malignant human skin cells (Hs27 fibroblasts, HaCaT keratinocytes, primary melanocytes) undergoing the aurin-induced heat shock response without impairment of viability. Aurin-induced melanoma cell apoptosis depends on Noxa up-regulation as confirmed by siRNA rescue experiments demonstrating that siPMAIP1-based target down-regulation suppresses aurin-induced cell death. Taken together, our data suggest feasibility of apoptotic elimination of malignant melanoma cells using the quinone methide-derived heat shock response inducer aurin.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Apoptose , Ácido Aurintricarboxílico/análogos & derivados , Proteínas de Choque Térmico/metabolismo , Melanoma/tratamento farmacológico , Neoplasias Cutâneas/tratamento farmacológico , Proteínas Adaptadoras de Transdução de Sinal , Ácido Aurintricarboxílico/química , Linhagem Celular Tumoral , Sobrevivência Celular , Ensaios de Seleção de Medicamentos Antitumorais , Citometria de Fluxo , Glutationa/metabolismo , Resposta ao Choque Térmico/genética , Humanos , Indolquinonas/química , Concentração Inibidora 50 , Queratinócitos/efeitos dos fármacos , Melanócitos/efeitos dos fármacos , Potencial da Membrana Mitocondrial , Modelos Moleculares , Estresse Oxidativo , Reação em Cadeia da Polimerase , RNA Interferente Pequeno/metabolismo , Regulação para CimaRESUMO
Cutaneous exposure to solar ultraviolet radiation (UVR) is a causative factor in photoaging and photocarcinogenesis. In human skin, oxidative stress is widely considered a key mechanism underlying the detrimental effects of acute and chronic UVR exposure. The lipid peroxidation product malondialdehyde (MDA) accumulates in tissue under conditions of increased oxidative stress, and the occurrence of MDA-derived protein epitopes, including dihydropyridine-lysine (DHP), has recently been substantiated in human skin. Here we demonstrate for the first time that acute exposure to sub-apoptogenic doses of solar simulated UV light (SSL) causes the formation of free MDA and protein-bound MDA-derived epitopes in cultured human HaCaT keratinocytes and healthy human skin. Immunohistochemical staining revealed that acute exposure to SSL is sufficient to cause an almost twenty-fold increase in general MDA- and specific DHP-epitope content in human skin. When compared to dose-matched solar simulated UVA, complete SSL was more efficient generating both free MDA and MDA-derived epitopes. Subsequent tissue microarray (TMA) analysis revealed the prevalence of MDA- and DHP-epitopes in nonmelanoma skin cancer (NMSC). In squamous cell carcinoma tissue, both MDA- and DHP-epitopes were increased more than threefold as compared to adjacent normal tissue. Taken together, these date demonstrate the occurrence of MDA-derived epitopes in both solar UVR-exposed healthy human skin and NMSC TMA tissue; however, the potential utility of these epitopes as novel biomarkers of cutaneous photodamage and a functional role in the process of skin photocarcinogenesis remain to be explored.