RESUMO
Mitochondrial Ca(2+) (Ca(2+)(m)) uptake is mediated by an inner membrane Ca(2+) channel called the uniporter. Ca(2+) uptake is driven by the considerable voltage present across the inner membrane (ΔΨ(m)) generated by proton pumping by the respiratory chain. Mitochondrial matrix Ca(2+) concentration is maintained five to six orders of magnitude lower than its equilibrium level, but the molecular mechanisms for how this is achieved are not clear. Here, we demonstrate that the mitochondrial protein MICU1 is required to preserve normal [Ca(2+)](m) under basal conditions. In its absence, mitochondria become constitutively loaded with Ca(2+), triggering excessive reactive oxygen species generation and sensitivity to apoptotic stress. MICU1 interacts with the uniporter pore-forming subunit MCU and sets a Ca(2+) threshold for Ca(2+)(m) uptake without affecting the kinetic properties of MCU-mediated Ca(2+) uptake. Thus, MICU1 is a gatekeeper of MCU-mediated Ca(2+)(m) uptake that is essential to prevent [Ca(2+)](m) overload and associated stress.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Sobrevivência Celular , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Apoptose , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/genética , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/genética , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Proteínas de Transporte da Membrana Mitocondrial/química , Proteínas de Transporte da Membrana Mitocondrial/genética , Membranas Mitocondriais/metabolismoRESUMO
Leukemogenesis is a complex process that involves multiple stages of mutation in either hematopoietic stem or progenitor cells, leading to cancer development over time. Acute myeloid leukemia (AML) is an aggressive malignancy that affects myeloid cells. The major disease burden is caused by immature blast cells, which are eliminated using conventional chemotherapies. Unfortunately, relapse is a leading cause of death in AML patients, with 30%-80% experiencing it within 2 years of initial treatment. The dominant cause of relapse in leukemia is the presence of therapy-resistant leukemic stem cells (LSCs). These cells express genes related to stemness that are frequently difficult to eradicate and tend to survive standard treatments. Studies have demonstrated that by targeting the metabolic pathways of LSCs, it is possible to improve outcomes and extend the survival of those afflicted by leukemia. The overwhelming evidence suggests that lipid metabolism is reprogrammed in LSCs, leading to an increase in fatty acid uptake and de novo lipogenesis. Genes regulating this process also play a crucial role in therapy evasion. In this concise review, we summarize the lipid metabolism in normal hematopoietic cells, AML blast cells, and AML LSCs. We also compare the lipid metabolic signatures in de novo versus therapy-resistant AML blast and LSCs. We further discuss the metabolic switches, cellular crosstalk, potential targets, and inhibitors of lipid metabolism that could alleviate treatment resistance and relapse.
Assuntos
Leucemia Mieloide Aguda , Células-Tronco Neoplásicas , Humanos , Células-Tronco Neoplásicas/metabolismo , Leucemia Mieloide Aguda/patologia , Carcinogênese/patologia , Recidiva , Lipídeos/uso terapêuticoRESUMO
BACKGROUND: Exposure to environmental tobacco smoke (ETS) is arguably the most ubiquitous and hazardous, even at very low levels, starting in early life. The objective of this study was to describe the state of research and future trends on ETS exposure and Children's Health (CH) topics with bibliometrics and altmetrics. METHODS: An electronic search was performed in Scopus database on January 31, 2023. Consensus was arrived on 100 most-cited articles by two reviewers. These papers were then cross matched with citations harvested from Web of Science (WoS) and Google Scholar. Altmetric Attention Score (AAS) and Dimension counts were also collected. Analysis and network visualization of authors, countries, and keywords were generated using VOSviewer software. RESULTS: Among a total of 1107 articles published on ETS and CH, the 100 top-cited articles appeared in 54 journals, with Pediatrics (n = 12) contributing a maximum number of articles. The time period between 2000 and 2009 accounted for 44% of all publications. With respect to the research design employed across these studies, cross-sectional design took precedence over others accounting for approximately 40%. Predominantly, articles focused on childhood asthma; however, current research trends have shifted towards emerging fields such as children's oral health and DNA methylation. Twitter, policy documents, and news outlets were the main platforms where outputs were discussed. The AAS was not associated with journal impact factor or access type. Weak correlations were observed between AAS and citation count in Scopus, WoS, and Google Scholar (r = 0.17 to 0.27) while a positive association existed between dimension count and the number of citations across all three databases (r = 0.84 to 0.98). CONCLUSION: This study demonstrates the evolution, digital dissemination and research hotspots in the field of ETS and CH, predicting the possible future research directions. High-quality studies with more specific exposure classification are warranted to better understand the relationship between ETS and CH.
Assuntos
Poluição por Fumaça de Tabaco , Humanos , Criança , Saúde da Criança , Estudos Transversais , Bibliometria , Fator de Impacto de RevistasRESUMO
BACKGROUND: Recent studies have emphasised the important role of amino acids in cancer metabolism. Cold physical plasma is an evolving technology employed to target tumour cells by introducing reactive oxygen species (ROS). However, limited understanding is available on the role of metabolic reprogramming in tumour cells fostering or reducing plasma-induced cancer cell death. METHODS: The utilisation and impact of major metabolic substrates of fatty acid, amino acid and TCA pathways were investigated in several tumour cell lines following plasma exposure by qPCR, immunoblotting and cell death analysis. RESULTS: Metabolic substrates were utilised in Panc-1 and HeLa but not in OVCAR3 and SK-MEL-28 cells following plasma treatment. Among the key genes governing these pathways, ASCT2 and SLC3A2 were consistently upregulated in Panc-1, Miapaca2GR, HeLa and MeWo cells. siRNA-mediated knockdown of ASCT2, glutamine depletion and pharmacological inhibition with V9302 sensitised HeLa cells to the plasma-induced cell death. Exogenous supplementation of glutamine, valine or tyrosine led to improved metabolism and viability of tumour cells following plasma treatment. CONCLUSION: These data suggest the amino acid influx driving metabolic reprogramming in tumour cells exposed to physical plasma, governing the extent of cell death. This pathway could be targeted in combination with existing anti-tumour agents.
Assuntos
Aminoácidos/metabolismo , Morte Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Neoplasias/metabolismo , Gases em Plasma/farmacologia , Argônio/farmacologia , Argônio/uso terapêutico , Células Cultivadas , Resistencia a Medicamentos Antineoplásicos/fisiologia , Metabolismo Energético/fisiologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Redes e Vias Metabólicas/efeitos dos fármacos , Redes e Vias Metabólicas/genética , Metaboloma/efeitos dos fármacos , Neoplasias/genética , Neoplasias/patologia , Neoplasias/terapia , Gases em Plasma/uso terapêutico , Espécies Reativas de Oxigênio/metabolismoRESUMO
Despite continuous advances in therapy, malignant melanoma is still among the deadliest types of cancer. At the same time, owing to its high plasticity and immunogenicity, melanoma is regarded as a model tumor entity when testing new treatment approaches. Cold physical plasma is a novel anticancer tool that utilizes a plethora of reactive oxygen species (ROS) being deposited on the target cells and tissues. To test whether plasma treatment would enhance the toxicity of an established antitumor therapy, ionizing radiation, we combined both physical treatment modalities targeting B16F10 murine melanoma cell in vitro. Repeated rather than single radiotherapy, in combination with gas plasma-introduced ROS, induced apoptosis and cell cycle arrest in an additive fashion. In tendency, gas plasma treatment sensitized the cells to subsequent radiotherapy rather than the other way around. This was concomitant with increased levels of TNFα, IL6, and GM-CSF in supernatants. Murine JAWS dendritic cells cultured in these supernatants showed an increased expression of cell surface activation markers, such as MHCII and CD83. For PD-L1 and PD-L2, increased expression was observed. Our results are the first to suggest an additive therapeutic effect of gas plasma and radiotherapy, and translational tumor models are needed to develop this concept further.
Assuntos
Fatores Imunológicos/uso terapêutico , Melanoma Experimental/imunologia , Melanoma Experimental/radioterapia , Gases em Plasma/uso terapêutico , Animais , Apoptose , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Fatores Imunológicos/farmacologia , Camundongos , Gases em Plasma/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Transcriptional co-activator with PDZ-binding motif (TAZ) and Yes-associated protein (YAP) are critical transcriptional co-activators downstream of the Hippo pathway involved in the regulation of organ size, tissue regeneration, proliferation, and apoptosis. Recent studies suggested common and distinct functions of TAZ and YAP and their diverse impact under several pathological conditions. Here we report differential regulation of TAZ and YAP in response to oxidative stress. H2O2 exposure leads to increased stability and activation of TAZ but not of YAP. H2O2 induces reversible S-glutathionylation at conserved cysteine residues within TAZ. We further demonstrate that TAZ S-glutathionylation is critical for reactive oxygen species (ROS)-mediated, TAZ-dependent TEA domain transcription factor (TEAD) trans-activation. Lysophosphatidic acid, a physiological activator of YAP and TAZ, induces ROS elevation and, subsequently, TAZ S-glutathionylation, which promotes TAZ-mediated target gene expression. TAZ expression is essential for renal homeostasis in mice, and we identify basal TAZ S-glutathionylation in murine kidney lysates, which is elevated during ischemia/reperfusion injury in vivo This induced nuclear localization of TAZ and increased expression of connective tissue growth factor. These results describe a novel mechanism by which ROS sustains total cellular levels of TAZ. This preferential regulation suggests TAZ to be a redox sensor of the Hippo pathway.
Assuntos
Cisteína/metabolismo , Glutationa/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Traumatismo por Reperfusão/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting , Proteínas de Ciclo Celular , Células Cultivadas , Fator de Crescimento do Tecido Conjuntivo/genética , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Cisteína/química , Glutationa/química , Via de Sinalização Hippo , Peróxido de Hidrogênio/farmacologia , Técnicas Imunoenzimáticas , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Proteínas Nucleares/genética , Oxidantes/farmacologia , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Transdução de Sinais/efeitos dos fármacos , Transativadores/genética , Fatores de Transcrição/genética , Proteínas com Motivo de Ligação a PDZ com Coativador TranscricionalRESUMO
Intracellular calcium overload plays a critical role in numerous pathological syndromes such as heart failure, brain ischemia, and stroke. Hyperactivation of the acid-sensing ion channels including degenerin/epithelial amiloride-sensitive sodium (DEG/ENaC) channels has been shown to elevate intracellular calcium and cause subsequent neuronal cell death that is independent of the canonical Egl-1/Ced-9/Ced-4/Ced-3 apoptotic pathway in Caenorhabditis elegans. In mammalian cells, hyperactivation of the DEG/ENaC channels can also lead to cell death, although the underlying mechanism remains largely unknown. Here, we use a tetracycline-inducible system to express the hyperactivation mutant of a mammalian DEG/ENaC channel protein, MDEG G430F, in murine kidney epithelial cells deficient in the key mitochondrial apoptotic proteins Bax and Bak. Remarkably, expression of MDEG G430F induces increased intracellular calcium, reactive oxygen species (ROS) production, and cell death. The MDEG G430F-induced cell death is blocked by the intracellular calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (acetoxymethyl ester), ROS scavengers, and the caspase inhibitor z-VAD-fmk (where z and fmk are benzyloxycarbonyl and fluoromethyl ketone). Mechanistically, the intracellular calcium overload and ROS increase lead to the inhibition of proteasomal and autophagic protein degradation, which promotes the accumulation of protein aggregates containing caspase-8 and subsequent caspase-8 activation. As protein aggregation upon the inhibition of proteasomal and autophagic degradation pathways is mediated by the ubiquitin-binding protein SQSTM1/p62 and the autophagy-related protein LC3, silencing of p62 and LC3 protects cells from MDEG G430F-induced cell death. Our results uncover a new mechanism of caspase-8-mediated apoptosis induced by intracellular calcium overload that is dependent on the autophagy-related proteins LC3 and p62 upon hyperactivation of DEG/ENaC channels.
Assuntos
Canais Iônicos Sensíveis a Ácido/metabolismo , Apoptose , Caspase 8/metabolismo , Ativação do Canal Iônico , Mamíferos/metabolismo , Substituição de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Caspase 8/química , Ativação Enzimática/efeitos dos fármacos , Células HEK293 , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas Mutantes/metabolismo , Poliubiquitina/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Ligação Proteica/efeitos dos fármacos , Estrutura Quaternária de Proteína , Ratos , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição TFIIH , Fatores de Transcrição/metabolismoRESUMO
The Ca(2+)-sensing stromal interaction molecule (STIM) proteins are crucial Ca(2+) signal coordinators. Cre-lox technology was used to generate smooth muscle (sm)-targeted STIM1-, STIM2-, and double STIM1/STIM2-knockout (KO) mouse models, which reveal the essential role of STIM proteins in Ca(2+) homeostasis and their crucial role in controlling function, growth, and development of smooth muscle cells (SMCs). Compared to Cre(+/-) littermates, sm-STIM1-KO mice showed high mortality (50% by 30 d) and reduced bodyweight. While sm-STIM2-KO was without detectable phenotype, the STIM1/STIM double-KO was perinatally lethal, revealing an essential role of STIM1 partially rescued by STIM2. Vascular and intestinal smooth muscle tissues from sm-STIM1-KO mice developed abnormally with distended, thinned morphology. While depolarization-induced aortic contraction was unchanged in sm-STIM1-KO mice, α1-adrenergic-mediated contraction was 26% reduced, and store-dependent contraction almost eliminated. Neointimal formation induced by carotid artery ligation was suppressed by 54%, and in vitro PDGF-induced proliferation was greatly reduced (79%) in sm-STIM1-KO. Notably, the Ca(2+) store-refilling rate in STIM1-KO SMCs was substantially reduced, and sustained PDGF-induced Ca(2+) entry was abolished. This defective Ca(2+) homeostasis prevents PDGF-induced NFAT activation in both contractile and proliferating SMCs. We conclude that STIM1-regulated Ca(2+) homeostasis is crucial for NFAT-mediated transcriptional control required for induction of SMC proliferation, development, and growth responses to injury.-Mancarella, S., Potireddy, S., Wang, Y., Gao, H., Gandhirajan, K., Autieri, M., Scalia, R., Cheng, Z., Wang, H., Madesh, M., Houser, S. R., Gill, D. L. Targeted STIM deletion impairs calcium homeostasis, NFAT activation, and growth of smooth muscle.
Assuntos
Cálcio/metabolismo , Proliferação de Células , Homeostase/fisiologia , Glicoproteínas de Membrana/metabolismo , Músculo Liso Vascular/metabolismo , Fatores de Transcrição NFATC/metabolismo , Animais , Canais de Cálcio , Deleção de Genes , Homeostase/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Intestinos/citologia , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Knockout , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Músculo Liso Vascular/citologia , Fatores de Transcrição NFATC/genética , Neointima/genética , Neointima/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , Molécula 1 de Interação Estromal , Molécula 2 de Interação Estromal , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/fisiologiaRESUMO
Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative condition, triggered by various factors causing the degeneration of upper and lower motor neurons, resulting in progressive muscle wasting, paralysis, and death. Multiple in vivo and in vitro models have been established to unravel the molecular events leading to the deterioration of motor neurons in ALS. The canonical and non-canonical Wnt signaling pathway has been implicated to play a crucial role in the progression of neurodegenerative disorders. This review discusses the role of Wnt signaling in the reported causes of ALS such as oxidative stress, mitochondrial dysfunction, autophagy, and apoptosis. Mutations in ALS-associated genes such as SOD1, C9orf72, TDP43, FUS, and OPTN cause an imbalance in neuronal integrity and homeostasis leading to motor neuron demise. Wnt signaling is also observed to play a crucial role in the muscle sparing of oculomotor neurons. The non-canonical Wnt/Ca2+ pathway which regulates intrinsic electrophysiological properties and mobilizes calcium ions to maintain neuronal integrity has been found to be altered in the stem cell-derived ALS model. Thus, the interplay of dysregulated canonical and non-canonical Wnt pathways in multiple motor neuron disease models has shown that Wnt contributes to disease progression indicating it to be utilized as a potential target for ALS.
Assuntos
Esclerose Lateral Amiotrófica , Humanos , Animais , Esclerose Lateral Amiotrófica/metabolismo , Via de Sinalização Wnt , Neurônios Motores/metabolismo , Estresse Oxidativo , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Modelos Animais de DoençasRESUMO
Ovarian cancers are tumors that originate from the different cells of the ovary and account for almost 4% of all the cancers in women globally. More than 30 types of tumors have been identified based on the cellular origins. Epithelial ovarian cancer (EOC) is the most common and lethal type of ovarian cancer which can be further divided into high-grade serous, low-grade serous, endometrioid, clear cell, and mucinous carcinoma. Ovarian carcinogenesis has been long attributed to endometriosis which is a chronic inflammation of the reproductive tract leading to progressive accumulation of mutations. Due to the advent of multi-omics datasets, the consequences of somatic mutations and their role in altered tumor metabolism has been well elucidated. Several oncogenes and tumor suppressor genes have been implicated in the progression of ovarian cancer. In this review, we highlight the genetic alterations undergone by the key oncogenes and tumor suppressor genes responsible for the development of ovarian cancer. We also summarize the role of these oncogenes and tumor suppressor genes and their association with a deregulated network of fatty acid, glycolysis, tricarboxylic acid and amino acid metabolism in ovarian cancers. Identification of genomic and metabolic circuits will be useful in clinical stratification of patients with complex etiologies and in identifying drug targets for personalized therapies against cancer.
RESUMO
Oxidation-reduction reactions played a significant role in the chemical evolution of life forms on oxygenated earth. Cellular respiration is dependent on such redox reactions, and any imbalance leads to the accumulation of reactive oxygen species (ROS), resulting in both chronic and acute illnesses. According to the International Agency for Research on Cancer (IARC), by 2040, the global burden of new cancer cases is expected to be around 27.5 million, with 16.3 million cancer deaths due to an increase in risk factors, such as unhealthy lifestyle, environmental factors, aberrant gene mutations, and resistance to therapies. ROS play an important role in cellular signaling, but they can cause severe damage to tissues when present at higher levels. Elevated and chronic levels of ROS are pertinent in carcinogenesis, while several therapeutic strategies rely on altering cellular ROS to eliminate tumor cells as they are more susceptible to ROS-induced damage than normal cells. Given this selective targeting potential, therapies that can effectively modulate ROS levels have been the focus of intense research in recent years. This review describes biologically relevant ROS, its origins in solid and hematological cancers, and the current status of evolving antioxidant and pro-oxidant therapies in cancers.
Assuntos
Neoplasias , Humanos , Espécies Reativas de Oxigênio/uso terapêutico , Neoplasias/tratamento farmacológico , Oxirredução , Antioxidantes/metabolismo , Carcinogênese , Estresse OxidativoRESUMO
Chronic lymphocytic leukemia (CLL) cells feature a pronounced apoptotic resistance. The vascular endothelial growth factor (VEGF) possesses a role in this apoptotic block, although underlying functional mechanisms and the involvement of the microenvironment are unclear. In this study, the VEGF status in CLL was assessed by enzyme-linked immunosorbent assay and immunofluorescence. VEGF receptor 2 (VEGFR2) phosphorylation was determined flow cytometrically and by immunofluorescence. For co-culture, CLL cells were cultivated on a monolayer of the bone marrow-derived stromal cell (BMSC) line HS5. Secreted VEGF was neutralized using the monoclonal antibody mAb293 (R&D Systems, Minneapolis, MN, USA). To block protein secretion, we used Brefeldin A. VEGF was downregulated in BMSCs by small interfering RNA (siRNA), and we assessed survival by annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining. CLL cells express and secrete VEGF and possess phosphorylated VEGFR2. This positive VEGF status is not sufficient to prevent spontaneous apoptosis in vitro. Coculture with BMSCs, which secrete vast amounts of VEGF, maintains in vitro CLL cell survival. Blockage of secreted VEGF using the monoclonal antibody mAb293 significantly reduced the survival support for cocultured CLL cells. Both general blockage of protein secretion by Brefeldin A in BMSCs, but not in CLL cells, and siRNA-mediated downregulation of VEGF in BMSCs, significantly reduced the coculture-mediated survival support for CLL cells. It can be concluded that BMSC-derived proteins and VEGF, in particular, but not CLL cell-derived VEGF, is essentially involved in the coculture-mediated survival support for CLL cells. Hence, therapeutic targeting of VEGF signaling might be a promising approach to overcome the apoptotic resistance CLL cells feature within their natural microenvironment.
Assuntos
Apoptose , Células da Medula Óssea/metabolismo , Células Estromais/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Anticorpos Monoclonais/farmacologia , Células da Medula Óssea/citologia , Brefeldina A/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Resistencia a Medicamentos Antineoplásicos , Citometria de Fluxo , Imunofluorescência , Humanos , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Fosforilação , Inibidores da Síntese de Proteínas/farmacologia , Interferência de RNA , Células Estromais/citologia , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/imunologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Cold physical plasma is a partially ionized gas investigated as a new anticancer tool in selectively targeting cancer cells in monotherapy or in combination with therapeutic agents. Here, we investigated the intrinsic resistance mechanisms of tumor cells towards physical plasma treatment. When analyzing the dose-response relationship to cold plasma-derived oxidants in 11 human cancer cell lines, we identified four 'resistant' and seven 'sensitive' cell lines. We observed stable intracellular glutathione levels following plasma treatment only in the 'resistant' cell lines indicative of altered antioxidant mechanisms. Assessment of proteins involved in GSH metabolism revealed cystine-glutamate antiporter xCT (SLC7A11) to be significantly more abundant in the 'resistant' cell lines as compared to 'sensitive' cell lines. This decisive role of xCT was confirmed by pharmacological and genetic inhibition, followed by cold physical plasma treatment. Finally, microscopy analysis of ex vivo plasma-treated human melanoma punch biopsies suggested a correlation between apoptosis and basal xCT protein abundance. Taken together, our results demonstrate that xCT holds the potential as a biomarker predicting the sensitivity of tumor cells towards plasma treatment.
Assuntos
Sistema y+ de Transporte de Aminoácidos/genética , Sistema y+ de Transporte de Aminoácidos/metabolismo , Resistencia a Medicamentos Antineoplásicos , Melanoma/genética , Gases em Plasma/farmacologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Feminino , Regulação Neoplásica da Expressão Gênica , Glutationa/metabolismo , Células HeLa , Humanos , Masculino , Melanoma/metabolismo , Pessoa de Meia-Idade , Regulação para CimaRESUMO
Cutaneous squamous cell carcinoma (SCC) is the most prevalent cancer worldwide, increasing the cost of healthcare services and with a high rate of morbidity. Its etiology is linked to chronic ultraviolet (UV) exposure that leads to malignant transformation of keratinocytes. Invasive growth and metastasis are severe consequences of this process. Therapy-resistant and highly aggressive SCC is frequently fatal, exemplifying the need for novel treatment strategies. Cold physical plasma is a partially ionized gas, expelling therapeutic doses of reactive oxygen and nitrogen species that were investigated for their anticancer capacity against SCC in vitro and SCC-like lesions in vivo. Using the kINPen argon plasma jet, a selective growth-reducing action of plasma treatment was identified in two SCC cell lines in 2D and 3D cultures. In vivo, plasma treatment limited the progression of UVB-induced SSC-like skin lesions and dermal degeneration without compromising lesional or non-lesional skin. In lesional tissue, this was associated with a decrease in cell proliferation and the antioxidant transcription factor Nrf2 following plasma treatment, while catalase expression was increased. Analysis of skin adjacent to the lesions and determination of global antioxidant parameters confirmed the local but not systemic action of the plasma anticancer therapy in vivo.
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In a significant proportion of acute myeloid leukemia (AML) cases the canonical WNT pathway is upregulated and targeting the WNT/LEF1 signaling cascade in AML may be a promising approach to develop new treatments for this entity. Recently two compounds (CGP049090 and PFK115-584) have been identified, which specifically inhibit complexation of beta-catenin (CTNNB1) and lymphoid enhancer-binding factor 1 (LEF1) leading to transcriptional inactivation of LEF1 in colon carcinoma cell lines. To evaluate the effect of WNT inhibition utilizing theses compounds with regard to their effectivity in AML we treated the AML cell lines Kasumi-1 and HL-60, primary AML blasts and healthy peripheral blood mononuclear cells (PBMCs) with varying concentrations of both substances. Treatment with both compounds for 24 h resulted in a significant killing of AML cell lines and primary AML blasts with 50% effective concentration doses (EC(50)) within the submicromolar range. PBMCs were not significantly affected as indicated by EC(50)-values 100-fold higher than for AML cells. Cell kill was mediated by apoptosis as indicated by induction of caspases 3 and 7 and cleavage of poly(ADP-ribose) polymerase (PARP) upon treatment. Furthermore, we could show that both compounds substantially decrease expression of CTNNB1/LEF1 target genes c-myc, cyclin D1 and survivin, proofing the specificity of the substances. This was shown in both, AML cell lines and most of the tested primary samples. Our data demonstrate that targeting this pathway seems to be an innovative approach in the treatment of AML.
Assuntos
Apoptose/efeitos dos fármacos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Perileno/análogos & derivados , Transdução de Sinais/efeitos dos fármacos , Proteínas Wnt/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Masculino , Pessoa de Meia-Idade , Estrutura Molecular , Perileno/química , Perileno/farmacologia , Células Tumorais Cultivadas , Proteínas Wnt/metabolismo , beta Catenina/metabolismoRESUMO
Increasing numbers of cancer deaths worldwide demand for new treatment avenues. Cold physical plasma is a partially ionized gas expelling a variety of reactive oxygen and nitrogen species, which can be harnesses therapeutically. Plasmas and plasma-treated liquids have antitumor properties in vitro and in vivo. Yet, global response signatures to plasma treatment have not yet been identified. To this end, we screened eight human cancer cell lines to investigate effects of low-dose, tumor-static plasma-treated medium (PTM) on cellular activity, immune-modulatory properties, and transcriptional levels of 22 redox-related genes. With PTM, a moderate reduction of metabolic activity and modest modulation of chemokine/cytokine pattern and markers of immunogenic cell death was observed. Strikingly, the Nuclear factor (erythroid-derived 2)-like 2 (nrf2) target heme oxygenase 1 (hmox1) was upregulated in all cell lines 4 h post PTM-treatment. nrf2 was not changed, but its baseline expression inversely and significantly correlated with hmox1 expression after exposure to PTM. Besides awarding hmox1 a central role with plasma-derived oxidants, we present a transcriptional redox map of 22 targets and chemokine/cytokine secretion map of 13 targets across eight different human tumor cell lines of four tumor entities at baseline activity that are useful for future studies in this field.
RESUMO
Malignant melanoma is an aggressive cancer that develops drug resistance leading to poor prognosis. Efficient delivery of chemotherapeutic drugs to the tumor tissue remains a major challenge in treatment regimens. Using murine (B16) and human (SK-MEL-28) melanoma cells, we investigated traditional cytotoxic agents in combination with cold physical plasma-derived oxidants. We report synergistic cytotoxicity of doxorubicin and epirubicin, and additive toxicity of oxaliplatin with plasma exposure in coefficient of drug interaction analysis. The combination treatment led to an increased DNA damage response (increased phosphorylation of ATM, γ-H2AX foci, and micronuclei formation). There was also an enhanced secretion of immunogenic cell death markers ATP and CXCL10 in cell culture supernatants following combination treatment. The observed synergistic effects in tumor cells was due to enhanced intracellular doxorubicin accumulation via upregulation of the organic cationic transporter SLC22A16 by plasma treatment. The doxorubicin uptake was reversed by pretreating cells with antioxidants or calcium influx inhibitor BTP2. Endoribonuclease-prepared siRNAs (esiRNA)-mediated knockdown of SLC22A16 inhibited the additive cytotoxic effect in tumor cells. SK-MEL 28 and THP-1 monocytes co-culture led to greater THP-1 cell migration and SK-MEL-28 cytotoxicity when compared with controls. Taken together, we propose pro-oxidant treatment modalities to sensitize chemoresistant melanoma cells towards subsequent chemotherapy, which may serve as therapeutic strategy in combination treatment in oncology.
Assuntos
Antineoplásicos/farmacologia , Células Epiteliais/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Proteínas de Transporte de Cátions Orgânicos/genética , Gases em Plasma/farmacologia , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Quimiocina CXCL10/genética , Quimiocina CXCL10/metabolismo , Técnicas de Cocultura , Terapia Combinada , Doxorrubicina/farmacologia , Epirubicina/farmacologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Histonas/genética , Histonas/metabolismo , Humanos , Melanoma Experimental , Camundongos , Proteínas de Transporte de Cátions Orgânicos/agonistas , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Oxaliplatina/farmacologia , Células THP-1 , Vorinostat/farmacologiaRESUMO
Despite striking advances in the treatment of metastasized melanoma, the disease is often still fatal. Attention is therefore paid towards combinational regimens. Oxidants endogenously produced in mitochondria are currently targeted in pre-clinical and clinical studies. Cytotoxic synergism of mitochondrial cytochrome c oxidase (CcO) inhibition in conjunction with addition of exogenous oxidants in 2D and 3D melanoma cell culture models were examined. Murine (B16) and human SK-MEL-28 melanoma cells exposed to low-dose CcO inhibitors (potassium cyanide or sodium azide) or exogenous oxidants alone were non-toxic. However, we identified a potent cytotoxic synergism upon CcO inhibition and plasma-derived oxidants that led to rapid onset of caspase-independent melanoma cell death. This was mediated by mitochondrial dysfunction induced by superoxide elevation and ATP depletion. This observation was validated by siRNA-mediated knockdown of COX4I1 in SK-MEL-28 cells with cytotoxicity in the presence of exogenous oxidants. Similar effects were obtained with ADDA 5, a recently identified specific inhibitor of CcO activity showing low toxicity in vivo. Human keratinocytes were not affected by this combinational treatment, suggesting selective effects on melanoma cells. Hence, targeting mitochondrial CcO activity in conjunction with exogenous pro oxidant therapies may constitute a new and effective melanoma treatment modality.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Oxidantes/toxicidade , Gases em Plasma/química , Animais , Caspase 3/metabolismo , Caspase 7/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Queratinócitos/efeitos dos fármacos , Melanoma/patologia , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Modelos Biológicos , RNA Interferente Pequeno/metabolismoRESUMO
Metastatic melanoma is an aggressive and deadly disease. Therapeutic advance has been achieved by antitumor chemo- and radiotherapy. These modalities involve the generation of reactive oxygen and nitrogen species, affecting cellular viability, migration, and immunogenicity. Such species are also created by cold physical plasma, an ionized gas capable of redox modulating cells and tissues without thermal damage. Cold plasma has been suggested for anticancer therapy. Here, melanoma cell toxicity, motility, and immunogenicity of murine metastatic melanoma cells were investigated following plasma exposure in vitro. Cells were oxidized by plasma, leading to decreased metabolic activity and cell death. Moreover, plasma decelerated melanoma cell growth, viability, and cell cycling. This was accompanied by increased cellular stiffness and upregulation of zonula occludens 1 protein in the cell membrane. Importantly, expression levels of immunogenic cell surface molecules such as major histocompatibility complex I, calreticulin, and melanocortin receptor 1 were significantly increased in response to plasma. Finally, plasma treatment significantly decreased the release of vascular endothelial growth factor, a molecule with importance in angiogenesis. Altogether, these results suggest beneficial toxicity of cold plasma in murine melanomas with a concomitant immunogenicity of potential interest in oncology.
Assuntos
Membrana Celular/metabolismo , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Oxidantes/farmacologia , Gases em Plasma , Animais , Linhagem Celular Tumoral , Membrana Celular/patologia , Melanoma/patologia , Camundongos , Proteína da Zônula de Oclusão-1/metabolismoRESUMO
Podocytes are terminally differentiated cells of the kidney filtration barrier. They are subjected to physiological filtration pressure and considerable mechanical strain, which can be further increased in various kidney diseases. When injury causes cytoskeletal reorganization and morphological alterations of these cells, the filtration barrier may become compromised and allow proteins to leak into the urine (a condition called proteinuria). Using time-resolved proteomics, we showed that podocyte injury stimulated the activity of the transcriptional coactivator YAP and the expression of YAP target genes in a rat model of glomerular disease before the development of proteinuria. Although the activities of YAP and its ortholog TAZ are activated by mechanical stress in most cell types, injury reduced YAP and TAZ activity in cultured human and mouse podocyte cell lines grown on stiff substrates. Culturing these cells on soft matrix or inhibiting stress fiber formation recapitulated the damage-induced YAP up-regulation observed in vivo, indicating a mechanotransduction-dependent mechanism of YAP activation in podocytes. YAP overexpression in cultured podocytes increased the abundance of extracellular matrix-related proteins that can contribute to fibrosis. YAP activity was increased in mouse models of diabetic nephropathy, and the YAP target CTGF was highly expressed in renal biopsies from glomerular disease patients. Although overexpression of human YAP in mice induced mild proteinuria, pharmacological inhibition of the interaction between YAP and its partner TEAD in rats ameliorated glomerular disease and reduced damage-induced mechanosignaling in the glomeruli. Thus, perturbation of YAP-dependent mechanosignaling is a potential therapeutic target for treating some glomerular diseases.