RESUMO
The Russian sector of the arctic shelf is the longest in the world. Quite a lot of places of massive discharge of bubble methane from the seabed into the water column and further into the atmosphere were found there. This natural phenomenon requires an extensive complex of geological, biological, geophysical, and chemical studies. This article is devoted to aspects of the use of a complex of marine geophysical equipment applied in the Russian sector of the arctic shelf for the detection and study of areas of the water and sedimentary strata with increased saturation with natural gases, as well as a description of some of the results obtained. This complex contains a single-beam scientific high-frequency echo sounder and multibeam system, a sub-bottom profiler, ocean-bottom seismographs, and equipment for continuous seismoacoustic profiling and electrical exploration. The experience of using the above equipment and the examples of the results obtained in the Laptev Sea have shown that these marine geophysical methods are effective and of particular importance for solving most problems related to the detection, mapping, quantification, and monitoring of underwater gas release from the bottom sediments of the shelf zone of the arctic seas, as well as the study of upper and deeper geological roots of gas emission and their relationship with tectonic processes. Geophysical surveys have a significant performance advantage compared to any contact methods. The large-scale application of a wide range of marine geophysical methods is essential for a comprehensive study of the geohazards of vast shelf zones, which have significant potential for economic use.
RESUMO
The Arctic seas are now of particular interest due to their prospects in terms of hydrocarbon extraction, development of marine transport routes, etc. Thus, various geohazards, including those related to seismicity, require detailed studies, especially by instrumental methods. This paper is devoted to the ocean-bottom seismographs (OBS) based on broadband molecular-electronic transfer (MET) sensors and a deployment case study in the Laptev Sea. The purpose of the study is to introduce the architecture of several modifications of OBS and to demonstrate their applicability in solving different tasks in the framework of seismic hazard assessment for the Arctic seas. To do this, we used the first results of several pilot deployments of the OBS developed by Shirshov Institute of Oceanology of the Russian Academy of Sciences (IO RAS) and IP Ilyinskiy A.D. in the Laptev Sea that took place in 2018-2020. We highlighted various seismological applications of OBS based on broadband MET sensors CME-4311 (60 s) and CME-4111 (120 s), including the analysis of ambient seismic noise, registering the signals of large remote earthquakes and weak local microearthquakes, and the instrumental approach of the site response assessment. The main characteristics of the broadband MET sensors and OBS architectures turned out to be suitable for obtaining high-quality OBS records under the Arctic conditions to solve seismological problems. In addition, the obtained case study results showed the prospects in a broader context, such as the possible influence of the seismotectonic factor on the bottom-up thawing of subsea permafrost and massive methane release, probably from decaying hydrates and deep geological sources. The described OBS will be actively used in further Arctic expeditions.
RESUMO
Our recent study demonstrated that sodium arsenite at a clinically relevant dose induced nephrotoxicity in human renal proximal tubular epithelial cell line HK-2, which could be inhibited by natural product 2,3,5,6-tetramethylpyrazine (TMP) with antioxidant activity. The present study demonstrated that arsenic exposure resulted in protein and enzymatic induction of heme oxygenase-1 (HO-1) in dose- and time-dependent manners in HK-2 cells. Blocking HO-1 enzymatic activity by zinc protoporphyrin (ZnPP) augmented arsenic-induced apoptosis, ROS production and mitochondrial dysfunction, suggesting a critical role for HO-1 as a renal protectant in this procession. On the other hand, TMP, upstream of HO-1, inhibited arsenic-induced ROS production and ROS-dependent HO-1 expression. TMP also prevented mitochondria dysfunction and suppressed activation of the intrinsic apoptotic pathway in HK-2 cells. Our results revealed that the regulation of arsenic-induced HO-1 expression was performed through multiple ROS-dependent signal pathways and the corresponding transcription factors, including p38 MAPK and JNK (but not ERK), AP-1, Nrf2 and NF-κB. TMP inhibited arsenic-induced activations of JNK, p38 MAPK, ERK, AP-1 and Nrf2 and block HO-1 protein expression. The present study, furthermore, demonstrated arsenic-induced expression of arsenic response protein 2 (ARS2) that was regulated by p38 MAPK, ERK and NF-κB. To our knowledge, this is the first report showing that ARS2 involved in arsenic-induced nephrotoxicity, while TMP pretreatment prevented such an up-regulation of ARS2 in HK-2 cells. Given ARS2 and HO-1 sharing the similar regulation mechanism, we speculated that ARS2 might also mediate cell survival in this procession. In summary, our study highlighted a role of HO-1 in the protection against arsenic-induced cytotoxicity downstream from the primary targets of TMP and further indicated that TMP may be used as a potential therapeutic agent in the treatment of arsenic-induced nephrotoxicity.
Assuntos
Antioxidantes/farmacologia , Arsenitos/toxicidade , Heme Oxigenase-1/metabolismo , Túbulos Renais Proximais/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , NF-kappa B/metabolismo , Proteínas Nucleares/metabolismo , Pirazinas/farmacologia , Compostos de Sódio/toxicidade , Fator de Transcrição AP-1/metabolismo , Apoptose/efeitos dos fármacos , Linhagem Celular , Citoproteção , Relação Dose-Resposta a Droga , Regulação para Baixo , Inibidores Enzimáticos/farmacologia , Heme Oxigenase-1/antagonistas & inibidores , Humanos , Túbulos Renais Proximais/enzimologia , Túbulos Renais Proximais/patologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de TempoRESUMO
During embryonic development, melanoblasts, the precursors of melanocytes, emerge from a subpopulation of the neural crest stem cells and migrate to colonize skin. Melanomas arise during melanoblast differentiation into melanocytes and from young proliferating melanocytes through somatic mutagenesis and epigenetic regulations. In the present study, we used several human melanoma cell lines from the sequential phases of melanoma development (radial growth phase, vertical growth phase and metastatic phase) to compare: (i) the frequency and efficiency of the induction of cell death via apoptosis and necroptosis; (ii) the presence of neural and cancer stem cell biomarkers as well as death receptors, DR5 and FAS, in both adherent and spheroid cultures of melanoma cells; (iii) anti-apoptotic effects of the endogenous production of cytokines and (iv) the ability of melanoma cells to perform neural trans-differentiation. We demonstrated that programed necrosis or necroptosis, could be induced in two metastatic melanoma lines, FEMX and OM431, while the mitochondrial pathway of apoptosis was prevalent in a vast majority of melanoma lines. All melanoma lines used in the current study expressed substantial levels of pluripotency markers, SOX2 and NANOG. There was a trend for increasing expression of Nestin, an early neuroprogenitor marker, during melanoma progression. Most of the melanoma lines, including WM35, FEMX and A375, can grow as a spheroid culture in serum-free media with supplements. It was possible to induce neural trans-differentiation of 1205Lu and OM431 melanoma cells in serum-free media supplemented with insulin. This was confirmed by the expression of neuronal markers, doublecortin and ß3-Tubulin, by significant growth of neurites and by the negative regulation of this process by a dominant-negative Rac1N17. These results suggest a relative plasticity of differentiated melanoma cells and a possibility for their neural trans-differentiation without the necessity for preliminary dedifferentiation.
Assuntos
Apoptose , Diferenciação Celular , Transdiferenciação Celular , Melanoma/metabolismo , Células-Tronco Neurais/metabolismo , Biomarcadores/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Células-Tronco Embrionárias/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Proteína Homeobox Nanog , Necrose , Nestina/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Receptor fas/metabolismoRESUMO
Although kidney is a target organ of arsenic cytotoxicity, the underlying mechanisms of arsenic-induced nephrotoxicity remain poorly understood. As tetramethylpyrazine (TMP) has recently been found to be a renal protectant in multiple kidney injuries, we hypothesize that TMP could suppress arsenic nephrotoxicity. In this study, human renal proximal tubular epithelial cell line HK-2 was used to elucidate the precise mechanisms of arsenic nephrotoxicity as well as the protective mechanism of TMP in these cells. Sodium arsenite exposure dramatically increased cellular reactive oxygen species (ROS) production, decreased levels of cellular glutathione (GSH), decreased cytochrome c oxidase activity and mitochondrial membrane potential, which indicated mitochondrial dysfunction. On the other hand, sodium arsenite activated pro-inflammatory signals, including ß-catenin, nuclear factor-κB (NF-κB), p38 mitogen-activated protein kinase (MAPK), tumor necrosis factor alpha and cyclooxygenase-2 (COX-2). Small molecule inhibitors of NF-κB and p38 MAPK blocked arsenic-induced COX-2 expression, suggesting arsenic-induced COX-2 up-regulation was NF-κB- and p38 MAPK-dependent. Finally, sodium arsenite induced autophagy in HK-2 cells at early phase (6 h) and the subsequent apoptosis at 24 h. Treatment by TMP or by the antioxidant N-acetylcysteine decreased arsenic-induced ROS production, enhanced GSH levels, prevented mitochondria dysfunction and suppressed the activation of pro-inflammatory signals and the development of autophagy and apoptosis. Our results suggested that TMP may be used as a new potential therapeutic agent to prevent arsenic-induced nephrotoxicity by suppressing these pathological processes.
Assuntos
Apoptose/efeitos dos fármacos , Arsenitos/toxicidade , Autofagia/efeitos dos fármacos , Mediadores da Inflamação/metabolismo , Nefropatias/induzido quimicamente , Túbulos Renais Proximais/química , Mitocôndrias/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Pirazinas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Compostos de Sódio/toxicidade , Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Proteínas Reguladoras de Apoptose/metabolismo , Biomarcadores/metabolismo , Linhagem Celular , Citoproteção , Relação Dose-Resposta a Droga , Glutationa/metabolismo , Humanos , Nefropatias/metabolismo , Nefropatias/patologia , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Fatores de TempoRESUMO
Ionizing radiation alone or in combination with chemotherapy is the main treatment modality for brain tumors including glioblastoma. Adult neurons and astrocytes demonstrate substantial radioresistance; in contrast, human neural stem cells (NSC) are highly sensitive to radiation via induction of apoptosis. Irradiation of tumor cells has the potential risk of affecting the viability and function of NSC. In this study, we have evaluated the effects of irradiated glioblastoma cells on viability, proliferation and differentiation potential of non-irradiated (bystander) NSC through radiation-induced signaling cascades. Using media transfer experiments, we demonstrated significant effects of the U87MG glioblastoma secretome after gamma-irradiation on apoptosis in non-irradiated NSC. Addition of anti-TRAIL antibody to the transferred media partially suppressed apoptosis in NSC. Furthermore, we observed a dramatic increase in the production and secretion of IL8, TGFß1 and IL6 by irradiated glioblastoma cells, which could promote glioblastoma cell survival and modify the effects of death factors in bystander NSC. While differentiation of NSC into neurons and astrocytes occurred efficiently with the corresponding differentiation media, pretreatment of NSC for 8 h with medium from irradiated glioblastoma cells selectively suppressed the differentiation of NSC into neurons, but not into astrocytes. Exogenous IL8 and TGFß1 increased NSC/NPC survival, but also suppressed neuronal differentiation. On the other hand, IL6 was known to positively affect survival and differentiation of astrocyte progenitors. We established a U87MG neurosphere culture that was substantially enriched by SOX2(+) and CD133(+) glioma stem-like cells (GSC). Gamma-irradiation up-regulated apoptotic death in GSC via the FasL/Fas pathway. Media transfer experiments from irradiated GSC to non-targeted NSC again demonstrated induction of apoptosis and suppression of neuronal differentiation of NSC. In summary, intercellular communication between glioblastoma cells and bystander NSC/NPC could be involved in the amplification of cancer pathology in the brain.
Assuntos
Apoptose/efeitos da radiação , Comunicação Celular/efeitos da radiação , Diferenciação Celular/efeitos da radiação , Células-Tronco Embrionárias/efeitos da radiação , Glioblastoma/metabolismo , Células-Tronco Neurais/efeitos da radiação , Transdução de Sinais/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Citocinas/metabolismo , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/patologia , Proteína Ligante Fas/metabolismo , Raios gama , Glioblastoma/patologia , Humanos , Ligantes , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Receptores de Morte Celular/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/metabolismoRESUMO
Adult neurons, which are terminally differentiated cells, demonstrate substantial radioresistance. In contrast, human neural stem cells (NSC), which have a significant proliferative capacity, are highly sensitive to ionizing radiation. Cranial irradiation that is widely used for treatment of brain tumors may induce death of NSC and further cause substantial cognitive deficits such as impairing learning and memory. The main goal of our study was to determine a mechanism of NSC radiosensitivity. We observed a constitutive high-level expression of TRAIL-R2 in human NSC. On the other hand, ionizing radiation through generation of reactive oxygen species targeted cell signaling pathways and dramatically changed the pattern of gene expression, including upregulation of TRAIL. A significant increase of endogenous expression and secretion of TRAIL could induce autocrine/paracrine stimulation of the TRAIL-R2-mediated signaling cascade with activation of caspase-3-driven apoptosis. Furthermore, paracrine stimulation could initiate bystander response of non-targeted NSC that is driven by death ligands produced by directly irradiated NSC. Experiments with media transfer from directly irradiated NSC to non-targeted (bystander) NSC confirmed a role of secreted TRAIL for induction of a death signaling cascade in non-targeted NSC. Subsequently, TRAIL production through elimination of bystander TRAIL-R-positive NSC might substantially restrict a final yield of differentiating young neurons. Radiation-induced TRAIL-mediated apoptosis could be partially suppressed by anti-TRAIL antibody added to the cell media. Interestingly, direct gamma-irradiation of SK-N-SH human neuroblastoma cells using clinical doses (2-5 Gy) resulted in low levels of apoptosis in cancer cells that was accompanied however by induction of a strong bystander response in non-targeted NSC. Numerous protective mechanisms were involved in the maintenance of radioresistance of neuroblastoma cells, including constitutive PI3K-AKT over-activation and endogenous synthesis of TGFß1. Specific blockage of these survival pathways was accompanied by a dramatic increase in radiosensitivity of neuroblastoma cells. Intercellular communication between cancer cells and NSC could potentially be involved in amplification of cancer pathology in the brain.
Assuntos
Apoptose/efeitos da radiação , Efeito Espectador/efeitos da radiação , Células-Tronco Neurais/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Linhagem Celular Tumoral , Raios gama/efeitos adversos , Humanos , Células-Tronco Neurais/patologia , Células-Tronco Neurais/efeitos da radiação , Neuroblastoma/patologia , Tolerância a Radiação , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Transdução de Sinais/efeitos da radiação , Ligante Indutor de Apoptose Relacionado a TNF/genéticaRESUMO
Chronic arsenic toxicity is a global health problem that affects more than 100 million people worldwide. Long-term health effects of inorganic sodium arsenite in drinking water may result in skin, lung and liver cancers and in severe neurological abnormalities. We investigated in the present study whether sodium arsenite affects signaling pathways that control cell survival, proliferation and neuronal differentiation of human neural stem cells (NSC). We demonstrated that the critical signaling pathway, which was suppressed by sodium arsenite in NSC, was the protective PI3K-AKT pathway. Sodium arsenite (2-4µM) also caused down-regulation of Nanog, one of the key transcription factors that control pluripotency and self-renewal of stem cells. Mitochondrial damage and cytochrome-c release induced by sodium arsenite exposure was followed by initiation of the mitochondrial apoptotic pathway in NSC. Beside caspase-9 and caspase-3 inhibitors, suppression of JNK activity decreased levels of arsenite-induced apoptosis in NSC. Neuronal differentiation of NSC was substantially inhibited by sodium arsenite exposure. Overactivation of JNK1 and ERK1/2 and down-regulation of PI3K-AKT activity induced by sodium arsenite were critical factors that strongly affected neuronal differentiation. In conclusion, sodium arsenite exposure of human NSC induces the mitochondrial apoptotic pathway, which is substantially accelerated due to the simultaneous suppression of PI3K-AKT. Sodium arsenite also negatively affects neuronal differentiation of NSC through overactivation of MEK-ERK and suppression of PI3K-AKT.
Assuntos
Apoptose , Arsenitos/farmacologia , Células-Tronco Neurais/efeitos dos fármacos , Neurogênese , Compostos de Sódio/farmacologia , Caspase 3/metabolismo , Caspase 8/metabolismo , Inibidores de Caspase/farmacologia , Proliferação de Células , Sobrevivência Celular , Cromonas/farmacologia , Citocromos c/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Imuno-Histoquímica , Sistema de Sinalização das MAP Quinases , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Morfolinas/farmacologia , Proteína Homeobox Nanog , Células-Tronco Neurais/enzimologia , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Transcrição GênicaRESUMO
Evolutionary potential of viruses can result in outbreaks of well-known viruses and emergence of novel ones. Pharmacological methods of intervening the reproduction of various less popular, but not less important viruses are not available, as well as the spectrum of antiviral activity for most known compounds. In the framework of chemical biology paradigm, characterization of antiviral activity spectrum of new compounds allows to extend the antiviral chemical space and provides new important structure-activity relationships for data-driven drug discovery. Here we present a primary assessment of antiviral activity of spiro-annulated derivatives of seven-membered heterocycles, oxepane and azepane, in phenotypic assays against viruses with different genomes, virion structures, and genome realization schemes: orthoflavivirus (tick-borne encephalitis virus, TBEV), enteroviruses (poliovirus, enterovirus A71, echovirus 30), adenovirus (human adenovirus C5), hantavirus (Puumala virus). Hit compounds inhibited reproduction of adenovirus C5, the only DNA virus in the studied set, in the yield reduction assay, and did not inhibit reproduction of RNA viruses.
Assuntos
Antivirais , Antivirais/farmacologia , Antivirais/química , Humanos , Compostos de Espiro/química , Compostos de Espiro/farmacologia , Relação Estrutura-Atividade , Oxepinas/química , Oxepinas/farmacologia , Animais , Replicação Viral/efeitos dos fármacos , FenótipoRESUMO
Sodium arsenite exposure at concentration >5 µM may induce embryotoxic and teratogenic effects in animal models. Long-term health effects of sodium arsenite from contaminated drinking water may result in different forms of cancer and neurological abnormalities. As cancer development processes seem to be originated in stem cells, we have chosen to examine the effects of sodium arsenite on signaling pathways and the corresponding transcription factors that regulate cell viability and self-renewal in mouse embryonic stem cells (ESC) and mouse neural stem/precursor cells. We demonstrated that the crucial signaling pathway, which was substantially suppressed by sodium arsenite exposure (4 µM) in ESC, was the PI3K-AKT pathway linked with numerous downstream targets that control cell survival and apoptosis. Furthermore, the whole core transcription factor circuitry that control self-renewal of mouse ESC (Stat3-P-Tyr705, Oct4, Sox2 and Nanog) was strongly down-regulated by sodium arsenite (4 µM) exposure. This was followed by G2/M arrest and induction of the mitochondrial apoptotic pathway that might be suppressed by caspase-9 and caspase-3 inhibitors. In contrast to mouse ESC with very low endogenous IL6, mouse neural stem/precursor cells (C17.2 clone immortalized by v-myc) with high endogenous production of IL6 exhibited a strong resistance to cytotoxic effects of sodium arsenite that could be decreased by inhibitory anti-IL6 antibody or Stat3 inhibition. In summary, our data demonstrated suppression of self-renewal and induction of apoptosis in mouse ESC by sodium arsenite exposure, which was further accelerated due to simultaneous inhibition of the protective PI3K-AKT and Stat3-dependent pathways.
Assuntos
Apoptose/efeitos dos fármacos , Arsenitos/farmacologia , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Transcrição STAT3/metabolismo , Compostos de Sódio/farmacologia , Animais , Sobrevivência Celular , Camundongos , Células-Tronco Neurais/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
Mitochondrial DNA depleted (ρ(0)) human skin fibroblasts (HSF) with suppressed oxidative phosphorylation were characterized by significant changes in the expression of 2100 nuclear genes, encoding numerous protein classes, in NF-κB and STAT3 signaling pathways, and by decreased activity of mitochondrial death pathway, compared to the parental ρ(+) HSF. In contrast, the extrinsic TRAIL/TRAIL-Receptor mediated death pathway remained highly active, and exogenous TRAIL in a combination with cycloheximide (CHX) induced higher levels of apoptosis in ρ(0) cells compared to ρ(+) HSF. Global gene expression analysis using microarray and qRT-PCR demonstrated that mRNA expression levels of many growth factors and their adaptor proteins (FGF13, HGF, IGFBP4, IGFBP6, and IGFL2), cytokines (IL6, ΙL17Β, ΙL18, ΙL19, and ΙL28Β) and cytokine receptors (IL1R1, IL21R, and IL31RA) were substantially decreased after mitochondrial DNA depletion. Some of these genes were targets of NF-κB and STAT3, and their protein products could regulate the STAT3 signaling pathway. Alpha-irradiation further induced expression of several NF-κB/STAT3 target genes, including IL1A, IL1B, IL6, PTGS2/COX2 and MMP12, in ρ(+) HSF, but this response was substantially decreased in ρ(0) HSF. Suppression of the IKK-NF-κB pathway by the small molecular inhibitor BMS-345541 and of the JAK2-STAT3 pathway by AG490 dramatically increased TRAIL-induced apoptosis in the control and irradiated ρ(+) HSF. Inhibitory antibodies against IL6, the main activator of JAK2-STAT3 pathway, added into the cell media, also increased TRAIL-induced apoptosis in HSF, especially after alpha-irradiation. Collectively, our results indicated that NF-κB activation was partially lost in ρ(0) HSF resulting in downregulation of the basal or radiation-induced expression of numerous NF-κB targets, further suppressing IL6-JAK2-STAT3 that in concert with NF-κB regulated protection against TRAIL-induced apoptosis.
Assuntos
Apoptose , Cicloeximida/farmacologia , DNA Mitocondrial/metabolismo , Raios gama , Perfilação da Expressão Gênica , NF-kappa B/metabolismo , Fator de Transcrição STAT3/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Biomarcadores/metabolismo , Western Blotting , Caspases/metabolismo , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/efeitos da radiação , Citometria de Fluxo , Imunofluorescência , Humanos , Luciferases/metabolismo , NF-kappa B/genética , Análise de Sequência com Séries de Oligonucleotídeos , Consumo de Oxigênio/efeitos dos fármacos , Consumo de Oxigênio/efeitos da radiação , Inibidores da Síntese de Proteínas/farmacologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT3/genética , Pele/citologia , Pele/efeitos dos fármacos , Pele/efeitos da radiaçãoRESUMO
Melanoma is the most lethal form of human skin cancer. However, only limited chemotherapy is currently available for the metastatic stage of the disease. Since chemotherapy, radiation and sodium arsenite treatment operate mainly through induction of the intrinsic mitochondrial pathway, a strongly decreased mitochondrial function in metastatic melanoma cells, could be responsible for low efficacy of the conventional therapy of melanoma. Another feature of metastatic melanoma cells is their proinflammatory phenotype, linked to endogenous expression of the inflammatory cytokines, such as TNFα IL6 and IL8, their receptors, and constitutive NF-κB- and STAT3-dependent gene expression, including cyclooxygenase-2 (PTGS2/COX2). In the present study, we treated melanoma cells with immunological (monoclonal antibody against TNFα or IL6), pharmacological (small molecular inhibitors of IKKß-NF-κB and JAK2-STAT3) or genetic (specific RNAi for COX-2) agents that suppressed the inflammatory response in combination with induction of apoptosis via TRAIL. As a result of these combined treatments, exogenous TRAIL via interactions with TRAIL-R2/R1 strongly increased levels of apoptosis in resistant melanoma cells. The present study provides new understanding of the regulation of TRAIL-mediated apoptosis in melanoma and will serve as the foundation for the potential development of a novel approach for a therapy of resistant melanomas.
Assuntos
Apoptose/efeitos dos fármacos , Melanoma/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Western Blotting , Linhagem Celular , Ciclo-Oxigenase 2/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , NF-kappa B/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Reação em Cadeia da Polimerase , Fator de Transcrição STAT3/metabolismoRESUMO
Treatment of melanoma cells by sodium arsenite or statins (simvastatin and lovastatin) dramatically modified activities of the main cell signaling pathways resulting in the induction of heme oxygenase-1 (HO-1) and in a downregulation of cyclooxygenase-2 (COX-2) protein levels. Through heme degradation and the production of carbon monoxide and biliverdin, HO-1 plays a protective role in different scenario of oxidative stress followed by mitochondrial apoptosis. Both sodium arsenite and statins could be efficient inducers of apoptosis in some melanoma cell lines, but often exhibited only modest proapoptotic activity in others, due to numerous protective mechanisms. We demonstrated in the present study that treatment by sodium arsenite or statins with an additional inhibition of HO-1 expression (or activation) caused a substantial upregulation of apoptosis in melanoma cells. Sodium arsenite- or statin-induced apoptosis was independent of BRAF status (wild type versus V600E) in melanoma lines. Monotreatment required high doses of statins (20-40 µM) for effective induction of apoptosis. As an alternative approach, pretreatment of melanoma cells with statin at decreased doses (5-20 µM) dramatically enhanced TRAIL-induced apoptosis, due to suppression of the NF-κB and STAT3-transcriptional targets (including COX-2) and downregulation of cFLIP-L (a caspase-8 inhibitor) protein levels. Furthermore, combined treatment with sodium arsenite and TRAIL or simvastatin and TRAIL efficiently induced apoptotic commitment in human neuroblastoma cells. In summary, our findings on enhancing effects of combined treatment of cancer cells using statin and TRAIL provide the rationale for further preclinical evaluation.
Assuntos
Aminoácidos/farmacologia , Apoptose/efeitos dos fármacos , Arsenitos/farmacologia , Melanoma/fisiopatologia , Neuroblastoma/fisiopatologia , Compostos de Sódio/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Linhagem Celular Tumoral , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Quimioterapia Combinada , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Humanos , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/metabolismo , Neuroblastoma/tratamento farmacológico , Neuroblastoma/genética , Neuroblastoma/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/farmacologiaRESUMO
Resistance of cancer cells to apoptosis is dependent on a balance of multiple genetic and epigenetic mechanisms, which up-regulate efficacy of the surviving growth factor-receptor signaling pathways and suppress death-receptor signaling pathways. The Insulin-like Growth Factor-1 Receptor (IGF-1R) signaling pathway is highly active in metastatic melanoma cells by mediating downstream activation of PI3K-AKT and MAPK pathways and controlling general cell survival and proliferation. In the present study, we used human melanoma lines with established genotypes that represented different phases of cancer development: radial-growth-phase WM35, vertical-growth-phase WM793, metastatic LU1205 and WM9 [1]. All these lines have normal NRAS. WM35, WM793, LU1205 and WM9 cells have mutated BRAF (V600E). WM35 and WM9 cells express normal PTEN, while in WM793 cells PTEN expression is down-regulated; finally, in LU1205 cells PTEN is inactivated by mutation. Cyclolignan picropodophyllin (PPP), a specific inhibitor of IGF-1R kinase activity, strongly down-regulated the basal levels of AKT activity in WM9 and in WM793 cells, modestly does so in LU1205, but has no effect on AKT activity in the early stage WM35 cells that are deficient in IGF-1R. In addition, PPP partially down-regulated the basal levels of active ERK1/2 in all lines used, highlighting the role of an alternative, non-BRAF pathway in MAPK activation. The final result of PPP treatment was an induction of apoptosis in WM793, WM9 and LU1205 melanoma cells. On the other hand, dose-dependent inhibition of IGF-1R kinase activity by PPP at a relatively narrow dose range (near 500 nM) has different effects on melanoma cells versus normal cells, inducing apoptosis in cancer cells and G2/M arrest of fibroblasts. To further enhance the pro-apoptotic effects of PPP on melanoma cells, we used a combined treatment of TNF-Related Apoptosis-Inducing Ligand (TRAIL) and PPP. This combination substantially increased death by apoptosis for WM793 and WM9 cells, but did so only modestly for LU1205 cells with very high basal activity of AKT. The ultimate goal of this direction of research is the discovery of a new treatment method for highly resistant human metastatic melanomas. Our findings provide the rationale for further preclinical evaluation of this novel treatment.
Assuntos
Apoptose/fisiologia , Melanoma/tratamento farmacológico , Receptor IGF Tipo 1/antagonistas & inibidores , Transdução de Sinais , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Sobrevivência Celular , Humanos , Melanoma/patologia , Podofilotoxina/análogos & derivados , Podofilotoxina/farmacologia , Podofilotoxina/uso terapêutico , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/genética , Células Tumorais CultivadasRESUMO
Radiotherapy combined with chemotherapy is the major treatment modality for human glioblastoma multiforme (GBM). GBMs eventually relapse after treatment and the average survival of GBM patients is less than two years. There is some evidence that cannabidiol (CBD) can induce cell death and increases the radiosensitivity of GBM by enhancing apoptosis. Beside initiation of death, CBD has been demonstrated as an inducer of autophagy. In the present study, we address the question whether CBD simultaneously induces a protective effect in GBM by upregulating autophagy. Addition of chloroquine that suppressed autophagic flux to 2D GBM cultures increased CBD-induced cell death, presenting proof for the protective autophagy. Blockage of autophagy upregulated radiation-induced cytotoxicity but only modestly affected the levels of cell death in CBD- or CBD/γ-irradiated 3D GBM cultures. Furthermore, CBD enhanced the pro-apoptotic activities of JNK1/2 and MAPK p38 signaling cascades while partially downregulated the pro-survival PI3K-AKT cascade, thereby changing a balance between cell death and survival. Suppression of JNK activation partially reduced CBD-induced cell death in 3D GBM cultures. In contrast, co-treatment of CBD-targeted cells with inhibitors of PI3K-AKT-NF-κB, IKK-NF-κB or JAK2-STAT3 pathways killed surviving GBM cells in both 2D and 3D cultures, potentially improving the therapeutic ratio of GBM.
Assuntos
Autofagia/efeitos dos fármacos , Neoplasias Encefálicas/tratamento farmacológico , Canabidiol/farmacologia , Glioblastoma/tratamento farmacológico , Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/genética , Glioblastoma/patologia , Glioblastoma/radioterapia , Humanos , Janus Quinase 2/genética , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/patologia , Fosfatidilinositol 3-Quinases/genética , Tolerância a Radiação/genética , Fator de Transcrição STAT3/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
Melanoma is the most lethal form of skin cancer. There is a lack of effective treatments for individuals with advanced disease. Many melanomas exhibit high levels of radioresistance. The direct consequence of gamma-irradiation for most melanoma cells is growth arrest at the G2-M phase of cell cycle. However, radiation-induced signaling pathways may affect numerous additional targets in cancer cells. We show in the present study that gamma-irradiation, as well as alpha-particle exposure, dramatically increases the susceptibility of melanoma cells to recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis via up-regulation of surface TRAIL-receptor 1/receptor 2 (DR4/DR5) levels and to Fas ligand-mediated apoptosis via up-regulation of surface Fas levels. Additionally, increased dynamin-2 expression after irradiation is critically important in the translocation of death receptor to the cell surface. Moreover, sodium arsenite treatment may up-regulate expression of endogenous TRAIL and induces its translocation to cell surface and further down-regulates cFLIP levels in melanoma cells. We have evaluated the effects of sequential gamma-irradiation and arsenite treatment of melanoma cells for the induction of death signaling. Such treatment results in an efficient TRAIL-mediated apoptosis via a paracrine mechanism. These data highlight the efficacy of combined modality treatment involving radiation and arsenite in clinical management of this often fatal form of skin cancer.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Arsenitos/farmacologia , Melanoma/tratamento farmacológico , Melanoma/radioterapia , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/radioterapia , Compostos de Sódio/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Partículas alfa , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Arsenitos/administração & dosagem , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Terapia Combinada , Cicloeximida/farmacologia , Sinergismo Farmacológico , Dinamina II/biossíntese , Dinamina II/metabolismo , Proteína Ligante Fas/biossíntese , Proteína Ligante Fas/metabolismo , Raios gama , Humanos , Melanoma/patologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/biossíntese , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Receptores do Fator de Necrose Tumoral/biossíntese , Receptores do Fator de Necrose Tumoral/metabolismo , Transdução de Sinais , Neoplasias Cutâneas/patologia , Compostos de Sódio/administração & dosagem , Ligante Indutor de Apoptose Relacionado a TNF/administração & dosagemRESUMO
Despite advances in glioblastoma (GBM) therapy, prognosis of the disease remains poor with a low survival rate. Cannabidiol (CBD) can induce cell death and enhance radiosensitivity of GBM but not normal astrocytes. Inhibition of ATM kinase is an alternative mechanism for radiosensitization of cancer cells. In this study, we increased the cytotoxic effects of the combination of CBD and γ-irradiation in GBM cells through additional inhibition of ATM kinase with KU60019, a small molecule inhibitor of ATM kinase. We observed in GBM cells treated by CBD, γ-irradiation and KU60019 high levels of apoptosis together with strong upregulation of the percentage of G2/M-arrested cells, blockade of cell proliferation and a massive production of pro-inflammatory cytokines. Overall, these changes caused both apoptotic and non-apoptotic inflammation-linked cell death. Furthermore, via JNK-AP1 activation in concert with active NF-κB, CBD upregulated gene and protein expression of DR5/TRAIL-R2 and sensitize GBM cells to TRAIL-induced apoptosis. In contrast, CBD notably decreased in GBM surface levels of PD-L1, a critical immune checkpoint agent for T-lymphocytes. We also used in the present study TS543 human proneural glioma cells that were grown as spheroid culture. TS543 neurospheres exhibited dramatic sensitivity to CBD-mediated killing that was additionally increased in combination with γ-irradiation and KU60019. In conclusion, treatment of human GBM by the triple combination (CBD, γ-irradiation and KU60019) could significantly increase cell death levels in vitro and potentially improve the therapeutic ratio of GBM.
RESUMO
[This corrects the article DOI: 10.18632/oncotarget.26582.].