RESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been responsible for the severe pandemic of acute respiratory disease, coronavirus disease 2019 (COVID-19), experienced in the 21st century. The clinical manifestations range from mild symptoms to abnormal blood coagulation and severe respiratory failure. In severe cases, COVID-19 manifests as a thromboinflammatory disease. Damage to the vascular compartment caused by SARS-CoV-2 has been linked to thrombosis, triggered by an enhanced immune response. The molecular mechanisms underlying endothelial activation have not been fully elucidated. We aimed to identify the proteins correlated to the molecular response of human umbilical vein endothelial cells (HUVECs) after exposure to SARS-CoV-2, which might help to unravel the molecular mechanisms of endothelium activation in COVID-19. In this direction, we exposed HUVECs to SARS-CoV-2 and analyzed the expression of specific cellular receptors, and changes in the proteome of HUVECs at different time points. We identified that HUVECs exhibit non-productive infection without cytopathic effects, in addition to the lack of expression of specific cell receptors known to be essential for SARS-CoV-2 entry into cells. We highlighted the enrichment of the protein SUMOylation pathway and the increase in SUMO2, which was confirmed by orthogonal assays. In conclusion, proteomic analysis revealed that the exposure to SARS-CoV-2 induced oxidative stress and changes in protein abundance and pathways enrichment that resembled endothelial dysfunction.
Assuntos
Fenômenos Biológicos , COVID-19 , Células Endoteliais , Humanos , Proteoma , Proteômica , SARS-CoV-2RESUMO
Non-coding RNAs (ncRNAs) comprise a diversity of RNA species, which do not have the potential to encode proteins. Non-coding RNAs include two classes of RNAs, namely: short regulatory ncRNAs and long non-coding RNAs (lncRNAs). The short regulatory RNAs, containing up to 200 nucleotides, include small RNAs, such as microRNAs (miRNA), short interfering RNAs (siRNAs), piwi-interacting RNAs (piRNAs), and small nucleolar RNAs (snoRNAs). The lncRNAs include long antisense RNAs and long intergenic RNAs (lincRNAs). Non-coding RNAs have been implicated as master regulators of several biological processes, their expression being strictly regulated under physiological conditions. In recent years, particularly in the last decade, substantial effort has been made to investigate the function of ncRNAs in several human diseases, including cancer. Glioblastoma is the most common and aggressive type of brain cancer in adults, with deregulated expression of small and long ncRNAs having been implicated in onset, progression, invasiveness, and recurrence of this tumor. The aim of this review is to guide the reader through important aspects of miRNA and lncRNA biology, focusing on the molecular mechanism associated with the progression of this highly malignant cancer type.
Assuntos
Regulação Neoplásica da Expressão Gênica , Predisposição Genética para Doença , Glioblastoma/genética , RNA não Traduzido/genética , Animais , Progressão da Doença , Estudos de Associação Genética/métodos , Glioblastoma/diagnóstico , Glioblastoma/terapia , Humanos , MicroRNAs/genética , Interferência de RNA , Estabilidade de RNA , RNA Longo não Codificante/genéticaRESUMO
BCL-X mRNA alternative splicing generates pro-apoptotic BCL-XS or anti-apoptotic BCL-XL gene products and the mechanism that regulates splice shifting is incompletely understood. We identified and characterized a long non-coding RNA (lncRNA) named INXS, transcribed from the opposite genomic strand of BCL-X, that was 5- to 9-fold less abundant in tumor cell lines from kidney, liver, breast and prostate and in kidney tumor tissues compared with non-tumors. INXS is an unspliced 1903 nt-long RNA, is transcribed by RNA polymerase II, 5'-capped, nuclear enriched and binds Sam68 splicing-modulator. Three apoptosis-inducing agents increased INXS lncRNA endogenous expression in the 786-O kidney tumor cell line, increased BCL-XS/BCL-XL mRNA ratio and activated caspases 3, 7 and 9. These effects were abrogated in the presence of INXS knockdown. Similarly, ectopic INXS overexpression caused a shift in splicing toward BCL-XS and activation of caspases, thus leading to apoptosis. BCL-XS protein accumulation was detected upon INXS overexpression. In a mouse xenograft model, intra-tumor injections of an INXS-expressing plasmid caused a marked reduction in tumor weight, and an increase in BCL-XS isoform, as determined in the excised tumors. We revealed an endogenous lncRNA that induces apoptosis, suggesting that INXS is a possible target to be explored in cancer therapies.
Assuntos
Apoptose , RNA Longo não Codificante/fisiologia , Proteína bcl-X/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Caspases/metabolismo , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Humanos , Neoplasias Renais/genética , Neoplasias Renais/patologia , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Isoformas de Proteínas/análise , Isoformas de Proteínas/genética , Splicing de RNA , RNA Longo não Codificante/análise , RNA Longo não Codificante/biossíntese , RNA Longo não Codificante/genética , Proteínas de Ligação a RNA/metabolismo , Proteína bcl-X/análise , Proteína bcl-X/genéticaRESUMO
In the original publication [...].
RESUMO
Introduction: Melanoma, a highly aggressive skin cancer originating in melanocytes, poses a significant threat due to its metastatic potential. While progress has been made in treating melanoma with targeted therapies and immunotherapies, challenges persist. Crotoxin (CTX), the principal toxin in Crotalus durissus terrificus snake venom, exhibits various biological activities, including anti-tumoral effects across multiple cancers. However, its clinical use is limited by toxicity. Thus, exploring alternatives to mitigate adverse effects is crucial. Methods and Results: This study investigates the antitumoral potential of CTX in its native and in a detoxified form, in melanoma cells. Firstly, we demonstrated that detoxified CTX presented reduced phospholipase activity. Both forms proved to be more cytotoxic to SK-MEL-28 and MeWo melanoma cells than non-tumoral cells. In SK-MEL-28 cells, where cytotoxic effects were more pronounced, native and detoxified CTX induced increased necrosis and apoptosis rates. We also confirmed the apoptosis death demonstrated by the activation of caspase-3 and 7, and the formation of apoptotic bodies. Furthermore, both CTX caused cell cycle arrest at the G2/M phase, interfering with melanoma cell proliferation. Cell migration and invasion were also suppressed by both CTX. These results confirm the antitumoral potential of CTX. Discussion: The maintenance of the antiproliferative effects in the detoxified version, with reduced enzymatic activity often liked to harm effects, supports further studies to identify active parts of the molecule responsible for the interesting effects without causing substantial toxic events, contributing to the future use of CTX-derived drugs with safety and efficacy.
RESUMO
Bothrops envenomation is a public health problem in Brazil. Despite the advances in the knowledge of the pathogenesis of systemic and local effects induced by Bothrops venom, the target tissues to this venom are not completely characterised. As preadipocytes are important cells of the adipose tissue and synthesize inflammatory mediators, we investigated the ability of B. moojeni snake venom (Bmv) to stimulate an inflammatory response in 3T3-L1 preadipocytes in vitro, focusing on (1) the release of PGE2, IL-6, TNF-α, MCP-1, KC, leptin and adiponectin; (2) the mechanisms involved in PGE2 release and (3) differentiation of these cells. Cytotoxicity of Bmv was determined by MTT assay. The concentrations of PGE2, cytokines and adipokines were quantified by EIA. Participation of the COX-1 and COX-2 enzymes, NF-κB and PGE2 receptors (EP1-4) was assessed using a pharmacological approach, and protein expression of the COX enzymes and P-NF-κB was analysed by western blotting. Preadipocyte differentiation was quantified by Oil Red O staining. Bmv (1 µg/mL) induced release of PGE2, IL-6 and KC and increased expression of COX-2 in preadipocytes. Basal levels of TNF-α, MCP-1, leptin and adiponectin were not modified. Treatment of cells with SC560 (COX-1 inhibitor) and NS398 (COX-2 inhibitor) inhibited Bmv-induced PGE2 release. Bmv induced phosphorylation of NF-κB, and treatment of the cells with TPCK and SN50, which inhibit distinct NF-κB domains, significantly reduced Bmv-induced PGE2 release, as did the treatment with an antagonist of PGE2 receptor EP1, unlike treatment with antagonists of EP2, EP3 or EP4. Bmv also induced lipid accumulation in differentiating cells. These results demonstrate that Bmv can activate an inflammatory response in preadipocytes by inducing the release of inflammatory mediators; that PGE2 production is mediated by the COX-1, COX-2 and NF-κB pathways; and that engagement of EP1 potentiates PGE2 synthesis via a positive feedback mechanism. Our findings highlight the role of the adipose tissue as another target for Bmv and suggest that it contributes to Bothrops envenomation by producing inflammatory mediators.
Assuntos
Bothrops , Adiponectina , Animais , Bothrops/metabolismo , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/metabolismo , Mediadores da Inflamação/metabolismo , Interleucina-6/metabolismo , Leptina , NF-kappa B , Venenos de Serpentes , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The pursuit of better therapies for disorders creating deficiencies in skeletal muscle regeneration is in progress, and several biotoxins are used in skeletal muscle research. Since recombinant proteins derived from Lonomia obliqua bristles, recombinant Lonomia obliqua Stuart-factor activator (rLosac) and recombinant Lonomia obliqua prothrombin activator protease (rLopap) act as cytoprotective agents and promote cell survival, we hypothesize that both rLosac and rLopap favour the skeletal muscle regeneration process. In the present work, we investigate the ability of these recombinant proteins rLosac and rLopap to modulate the production of key mediators of the myogenic process. The expression of myogenic regulatory factors (MRFs), cell proliferation, the production of prostaglandin E2 (PGE2) and the protein expression of cyclooxygenases COX-1 and COX-2 were evaluated in C2C12 mouse myoblasts pre-treated with rLosac and rLopap. We found an increased proliferation of myoblasts, stimulated by both recombinant proteins. Moreover, these proteins modulated PGE2 release and MRFs activities. We also found an increased expression of the EP4 receptor in the proliferative phase of C2C12 cells, suggesting the involvement of this receptor in the effects of PGE2 in these cells. Moreover, the recombinant proteins inhibited the release of IL-6 and PGE2, which is induced by an inflammatory stimulus by IL-1ß. This work reveals rLopap and rLosac as promising proteins to modulate processes involving tissue regeneration as occurs during skeletal muscle injury.
RESUMO
Salivary glands are vital structures responsible for successful tick feeding. The saliva of ticks contains numerous active molecules that participate in several physiological processes. A Kunitz-type factor Xa (FXa) inhibitor, similar to the tissue factor pathway inhibitor (TFPI) precursor, was identified in the salivary gland transcriptome of Amblyomma sculptum ticks. The recombinant mature form of this Kunitz-type inhibitor, named Amblyomin-X, displayed anticoagulant, antiangiogenic, and antitumor properties. Amblyomin-X is a protein that inhibits FXa in the blood coagulation cascade and acts via non-hemostatic mechanisms, such as proteasome inhibition. Amblyomin-X selectively induces apoptosis in cancer cells and promotes tumor regression through these mechanisms. Notably, the cytotoxicity of Amblyomin-X seems to be restricted to tumor cells and does not affect non-tumorigenic cells, tissues, and organs, making this recombinant protein an attractive molecule for anticancer therapy. The cytotoxic activity of Amblyomin-X on tumor cells has led to vast exploration into this protein. Here, we summarize the function, action mechanisms, structural features, pharmacokinetics, and biodistribution of this tick Kunitz-type inhibitor recombinant protein as a promising novel antitumor drug candidate.
RESUMO
Increased collagen-derived advanced glycation end-products (AGEs) are consistently related to painful diseases, including osteoarthritis, diabetic neuropathy, and neurodegenerative disorders. We have recently developed a model combining a two-dimensional glycated extracellular matrix (ECM-GC) and primary dorsal root ganglion (DRG) that mimicked a pro-nociceptive microenvironment. However, culturing primary cells is still a challenge for large-scale screening studies. Here, we characterized a new model using ECM-GC as a stimulus for human sensory-like neurons differentiated from SH-SY5Y cell lines to screen for analgesic compounds. First, we confirmed that the differentiation process induces the expression of neuron markers (MAP2, RBFOX3 (NeuN), and TUBB3 (ß-III tubulin), as well as sensory neuron markers critical for pain sensation (TRPV1, SCN9A (Nav1.7), SCN10A (Nav1.8), and SCN11A (Nav1.9). Next, we showed that ECM-GC increased c-Fos expression in human sensory-like neurons, which is suggestive of neuronal activation. In addition, ECM-GC upregulated the expression of critical genes involved in pain, including SCN9A and TACR1. Of interest, ECM-GC induced substance P release, a neuropeptide widely involved in neuroinflammation and pain. Finally, morphine, the prototype opiate, decreased ECM-GC-induced substance P release. Together, our results suggest that we established a functional model that can be useful as a platform for screening candidates for the management of painful conditions.
Assuntos
Analgésicos/análise , Analgésicos/farmacologia , Colágeno/farmacologia , Avaliação Pré-Clínica de Medicamentos , Modelos Biológicos , Células Receptoras Sensoriais/citologia , Animais , Antígenos de Neoplasias/metabolismo , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Galectina 3/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Glicosilação/efeitos dos fármacos , Humanos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.7/genética , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Neuritos/efeitos dos fármacos , Neuritos/metabolismo , Neurônios/citologia , Neurônios/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fos/metabolismo , Ratos , Receptores da Neurocinina-1/genética , Receptores da Neurocinina-1/metabolismo , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/metabolismo , Substância P/metabolismo , beta-Endorfina/metabolismoRESUMO
We identified Pycard and BC017158 genes as putative effectors of the Quantitative Trait locus (QTL) that we mapped at distal chromosome 7 named Irm1 for Inflammatory response modulator 1, controlling acute inflammatory response (AIR) and the production of IL-1ß, dependent on the activation of the NLRP3 inflammasome. We obtained the mapping through genome-wide linkage analysis of Single Nucleotide Polymorphisms (SNPs) in a cross between High (AIRmax) and Low (AIRmin) responder mouse lines that we produced by several generations of bidirectional selection for Acute Inflammatory Response. A highly significant linkage signal (LOD score peak of 72) for ex vivo IL-1ß production limited a 4 Mbp interval to chromosome 7. Sequencing of the locus region revealed 14 SNPs between "High" and "Low" responders that narrowed the locus to a 420 Kb interval. Variants were detected in non-coding regions of Itgam, Rgs10 and BC017158 genes and at the first exon of Pycard gene, resulting in an E19K substitution in the protein ASC (apoptosis associated speck-like protein containing a CARD) an adaptor molecule in the inflammasome complex. Silencing of BC017158 inhibited IL1-ß production by stimulated macrophages and the E19K ASC mutation carried by AIRmin mice impaired the ex vivo IL-1ß response and the formation of ASC specks in stimulated cells. IL-1ß and ASC specks play major roles in inflammatory reactions and in inflammation-related diseases. Our results delineate a novel genetic factor and a molecular mechanism affecting the acute inflammatory response.
Assuntos
Proteínas Adaptadoras de Sinalização CARD , Inflamassomos , Animais , Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Ligação Genética , Inflamassomos/genética , Inflamassomos/metabolismo , Inflamação/genética , Inflamação/metabolismo , Camundongos , Locos de Características QuantitativasRESUMO
Although crotoxin B (CB) is a well-established catalytically active secretory phospholipase A2 group IIA (sPLA2-IIA) myotoxin, we investigated its potential stimulatory effect on myogenesis with the involvement of prostaglandins (PGs) produced by cyclooxygenase (COX)-1 and -2 pathways. Myoblast C2C12 were cultured in proliferation or commitment protocols and incubated with CB followed by lumiracoxib (selective COX-2 inhibitor) or valeryl salicylate (selective COX-1 inhibitor) and subjected to analysis of PG release, cell proliferation and activation of myogenic regulatory factors (MRFs). Our data showed that CB in non-cytotoxic concentrations induces an increase of COX-2 protein expression and stimulates the activity of both COX isoforms to produce PGE2, PGD2 and 15d-PGJ2. CB induced an increase in the proliferation of C2C12 myoblast cells dependent on PGs from both COX-1 and COX-2 pathways. In addition, CB stimulated the activity of Pax7, MyoD, Myf5 and myogenin in proliferated cells. Otherwise, CB increased myogenin activity but not MyoD in committed cells. Our findings evidence the role of COX-1- and COX-2-derived PGs in modulating CB-induced activation of MRFs. This study contributes to the knowledge that CB promote early myogenic events via regulatory mechanisms on PG-dependent COX pathways, showing new concepts about the effect of sPLA2-IIA in skeletal muscle repair.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Crotoxina/farmacologia , Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 2/metabolismo , Fosfolipases A2 do Grupo II/farmacologia , Proteínas de Membrana/metabolismo , Desenvolvimento Muscular/efeitos dos fármacos , Mioblastos Esqueléticos/efeitos dos fármacos , Neurotoxinas/farmacologia , Prostaglandinas/metabolismo , Animais , Linhagem Celular , Camundongos , Proteína MyoD/metabolismo , Mioblastos Esqueléticos/enzimologia , Fator Regulador Miogênico 5/metabolismo , Miogenina/metabolismo , Fator de Transcrição PAX7/metabolismo , Transdução de SinaisRESUMO
Envenomation caused by contact with Lonomia obliqua bristles is characterized by pain, an intense systemic proinflammatory reaction and disturbances in the coagulation cascade that can cause severe clinical manifestations and death. However, the role of immune system components in these effects is still poorly understood. In this study, we evaluated the cytotoxic effect of L. obliqua venom on THP-1-derived macrophages and its ability to modulate inflammatory markers, as well as the cytokine and chemokine release profile. Our results show that L. obliqua venom is able to directly exert a potent pro-inflammatory reaction in macrophages, characterized by the activation of the NF-κB transcription factor pathway, the expression of CD80 and CD83, and the release of pro-inflammatory mediators such as TNF-α, IL-1ß, IL-6, IL-8 and CXCL10. These results suggest that macrophages can play an important role during the orchestration of the inflammatory response present in envenomation caused by Lonomia obliqua caterpillars.
Assuntos
Venenos de Artrópodes/toxicidade , Larva , Macrófagos/efeitos dos fármacos , NF-kappa B/metabolismo , Animais , Antígenos CD/metabolismo , Antígeno B7-1/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Citocinas/genética , Citocinas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Imunoglobulinas/metabolismo , Lepidópteros , Macrófagos/metabolismo , Glicoproteínas de Membrana/metabolismo , Células THP-1 , Antígeno CD83RESUMO
As a tribute to Butantan Institute in its 120th anniversary, this review describes some of the scientific research efforts carried out in the study of Lonomia envenoming in Brazil, a country where accidents with caterpillars reach over 42,000 individuals per year (especially in South and Southeast Brazil). Thus, the promising data regarding the studies with Lonomia's toxins contributed to the creation of new research centers specialized in toxinology based at Butantan Institute, as well as to the production of the antilonomic serum (ALS), actions which are in line with the Butantan Institute mission "to research, develop, manufacture, and provide products and services for the health of the population". In addition, the study of the components of the Lonomia obliqua bristle extract led to the discovery of new molecules with peculiar properties, opening a field of knowledge that could lead to the development and innovation of new drugs aimed at cell regeneration and inflammatory diseases.
Assuntos
Venenos de Artrópodes/toxicidade , Borboletas/fisiologia , Mordeduras e Picadas de Insetos/terapia , Animais , Brasil/epidemiologia , Humanos , Mordeduras e Picadas de Insetos/epidemiologia , Larva/fisiologiaRESUMO
In this study, we investigated the effects and mechanisms of the pro-inflammatory cytokines IL-1ß and TNF-α on the proliferation and commitment phases of myoblast differentiation. C2C12 mouse myoblast cells were cultured to reach a proliferated or committed status and were incubated with these cytokines for the evaluation of cell proliferation, cyclooxygenase 2 (COX-2) expression, release of prostaglandins (PGs) and myokines, and activation of myogenic regulatory factors (MRFs). We found that inhibition of the IL-6 receptor reduced IL-1ß- and TNF-α-induced cell proliferation, and that the IL-1ß effect also involved COX-2-derived PGs. Both cytokines modulated the release of the myokines myostatin, irisin, osteonectin, and IL-15. TNF-α and IL-6 reduced the activity of Pax7 in proliferated cells and reduced MyoD and myogenin activity at both proliferative and commitment stages. Otherwise, IL-1ß increased myogenin activity only in committed cells. Our data reveal a key role of IL-6 and COX-2-derived PGs in IL-1ß and TNF-α-induced myoblast proliferation and support the link between TNF-α and IL-6 and the activation of MRFs. We concluded that IL-1ß and TNF-α induce similar effects at the initial stages of muscle regeneration but found critical differences between their effects with the progression of the process, bringing new insights into inflammatory signalling in skeletal muscle regeneration.
Assuntos
Proliferação de Células/efeitos dos fármacos , Ciclo-Oxigenase 2/metabolismo , Interleucina-1beta/farmacologia , Interleucina-6/metabolismo , Mioblastos/metabolismo , Prostaglandinas/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Inibidores de Ciclo-Oxigenase 2/farmacologia , Diclofenaco/análogos & derivados , Diclofenaco/farmacologia , Camundongos , Desenvolvimento Muscular/efeitos dos fármacos , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Receptores de Interleucina-6/antagonistas & inibidores , Regeneração/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
OBJECTIVE: Cell growth curves constitute one of the primary assays employed to analyze cell proliferation dynamics of in vitro cultured cells under specific culture conditions. From the cell growth curve, it is possible to assess the behavior of proliferating cells under different conditions, such as drug treatment and genomic editions. Traditionally, growth curves for adherent cells are obtained by seeding the cells in multiple-well plates and counting the total number of cells at different time points. Here, we compare this traditional method to the fluorescence-based method, which is based on the CFSE fluorescence decay over time. RESULTS: The fluorescence-based method is not dependent on the determination of the total number of cells, but rather is approached by assessing the fluorescence of a sample of single cells from a cell population at different time points after plating. Therefore, this method is not biased due to either cell loss during harvesting or to the presence of cellular debris and cell clumps. Moreover, the fluorescence-based method displays lower variation among different measurements of the same time point, which increases the reliability on the determination of lag, log and stationary phase transitions.
Assuntos
Contagem de Células/métodos , Células/citologia , Adesão Celular , Proliferação de Células , Fluoresceínas/metabolismo , Fluorescência , Células HEK293 , Humanos , Succinimidas/metabolismoRESUMO
Pararamosis is a disease that occurs due to contact with the hairs of the larval stage of the Brazilian moth Premolis semirufa. Envenomation induces osteoarticular alterations with cartilage impairment that resembles joint synovitis. Thus, the toxic venom present in the caterpillar hairs interferes with the phenotype of the cells present in the joints, resulting in inflammation and promoting tissue injury. Therefore, to address the inflammatory mechanisms triggered by envenomation, we studied the effects of P. semirufa hair extract on human chondrocytes. We have selected for the investigation, cytokines, chemokines, matrix metalloproteinases (MMPs), complement components, eicosanoids, and extracellular matrix (ECM) components related to OA and RA. In addition, for measuring protein-coding mRNAs of some molecules associated with osteoarthritis (OA) and rheumatoid arthritis (RA), reverse transcription (RT) was performed followed by quantitative real-time PCR (RT-qPCR) and we performed the RNA-sequencing (RNA-seq) analysis of the chondrocytes transcriptome. In the supernatant of cell cultures treated with the extract, we observed increased IL-6, IL-8, MCP-1, prostaglandin E2, metalloproteinases (MMP-1, MMP-2, MMP-3 and MMP-13), and complement system components (C3, C4, and C5). We noticed a significant decrease in both aggrecan and type II collagen and an increase in HMGB1 protein in chondrocytes after extract treatment. RNA-seq analysis of the chondrocyte transcriptome allowed us to identify important pathways related to the inflammatory process of the disease, such as the inflammatory response, chemotaxis of immune cells and extracellular matrix (ECM) remodeling. Thus, these results suggest that components of Premolis semirufa hair have strong inflammatory potential and are able to induce cartilage degradation and ECM remodeling, promoting a disease with an osteoarthritis signature. Modulation of the signaling pathways that were identified as being involved in this pathology may be a promising approach to develop new therapeutic strategies for the control of pararamosis and other inflammatory joint diseases.
Assuntos
Cartilagem/patologia , Condrócitos/fisiologia , Inflamação/imunologia , Artropatias/imunologia , Osteoartrite/genética , Animais , Venenos de Artrópodes/metabolismo , Células Cultivadas , Citocinas/metabolismo , Matriz Extracelular/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Artropatias/induzido quimicamente , Mariposas/metabolismo , Floresta Úmida , Transdução de SinaisRESUMO
We have investigated Amblyomin-X-treated horse melanomas to better understand its mode of action through transcriptome analysis and the in vivo model. Amblyomin-X is a Kunitz-type homologous protein that selectively leads to the death of tumor cells via ER stress and apoptosis, currently under investigation as a new drug candidate for cancer treatment. Melanomas are immunogenic tumors, and a better understanding of the immune responses is warranted. Equine melanomas are spontaneous and not so aggressive as human melanomas are, as this study shows that the in vivo treatment of encapsulated horse melanoma tumors led to a significant reduction in the tumor size or even the complete disappearance of the tumor mass through intratumoral injections of Amblyomin-X. Transcriptome analysis identified ER- and mitochondria-stress, modulation of the innate immune system, apoptosis, and possibly immunogenic cell death activation. Interactome analysis showed that Amblyomin-X potentially interacts with key elements found in transcriptomics. Taken together, Amblyomin-X modulated the tumor immune microenvironment in different ways, at least contributing to induce tumor cell death.
Assuntos
Antineoplásicos/uso terapêutico , Proteínas de Artrópodes/uso terapêutico , Doenças dos Cavalos/tratamento farmacológico , Melanoma/veterinária , Proteínas e Peptídeos Salivares/uso terapêutico , Animais , Morte Celular/efeitos dos fármacos , Descoberta de Drogas , Cavalos , Masculino , Melanoma/tratamento farmacológico , Microambiente Tumoral/efeitos dos fármacosRESUMO
Long non-coding RNAs (lncRNAs) serve critical roles in regulating cellular homeostasis, and their deregulated expression/activity is associated with neoplastic transformation. The maternally expressed gene 3 (MEG3) has been extensively described as a tumor suppressor gene in different types of cancer, including breast cancer. Interestingly, using a panel of seven different breast cancer cell lines, the present study revealed that MEG3 is highly expressed in the triple negative metastatic human Hs578T breast cancer cell line, which is refractory to different therapeutic approaches. Therefore, the present study aimed to investigate the phenotypic impact of MEG3 deletion in this cell line. Using the CRISPR/Cas9 system, complete knockout (KO) of MEG3 was achieved. Deletion was confirmed by genomic PCR and reverse transcription-quantitative PCR. The MEG3_KO cell population displaying the highest efficiency of genomic editing was selected for phenotypic in vitro assays, including wound scratch and Transwell assays, flow cytometry and immunofluorescence. The results demonstrated that MEG3 deletion increased cell proliferation, anchorage-independent cell growth and cell motility, which was consistent with its well-known tumor suppressor function. However, the present study revealed that MEG3_KO also lead to decreased cell invasiveness ability, supporting previous evidence that MEG3 modulates epithelial-to-mesenchymal inducing factors. The present study demonstrated that deletion of MEG3 promoted an increase in transforming growth factor ß and N-cadherin protein levels and significant reduction in matrix metallopeptidase 2, zinc-finger E-box binding homeobox 1 and collagen type III α1 chain gene expression levels. Additionally, MEG3_KO cells displayed significant resistance to doxorubicin treatment, demonstrating the role of this lncRNA in cancer cell survival by regulating apoptosis. The present study highlighted the utility of CRISPR/Cas9 for anticancer studies of intergenic lncRNAs and demonstrated that, although Hs578T cells express MEG3 at high levels, these cells display mechanisms to escape the growth suppression effects of this lncRNA. Notably, the detailed pathological mechanisms of MEG3 concerning tumor metastasis remain to be elucidated prior to applying MEG3 expression/activation in future therapeutic approaches for breast cancer treatment.
RESUMO
Chromatin remodeler proteins exert an important function in promoting dynamic modifications in the chromatin architecture, performing a central role in regulating gene transcription. Deregulation of these molecular machines may lead to striking perturbations in normal cell function. The CHD7 gene is a member of the chromodomain helicase DNA-binding family and, when mutated, has been shown to be the cause of the CHARGE syndrome, a severe developmental human disorder. Moreover, CHD7 has been described to be essential for neural stem cells and it is also highly expressed or mutated in a number of human cancers. However, its potential role in glioblastoma has not yet been tested. Here, we show that CHD7 is up-regulated in human glioma tissues and we demonstrate that CHD7 knockout (KO) in LN-229 glioblastoma cells suppresses anchorage-independent growth and spheroid invasion in vitro. Additionally, CHD7 KO impairs tumor growth and increases overall survival in an orthotopic mouse xenograft model. Conversely, ectopic overexpression of CHD7 in LN-428 and A172 glioblastoma cell lines increases cell motility and invasiveness in vitro and promotes LN-428 tumor growth in vivo. Finally, RNA-seq analysis revealed that CHD7 modulates a specific transcriptional signature of invasion-related target genes. Further studies should explore clinical-translational implications for glioblastoma treatment.