RESUMO
BACKGROUND: Immunotherapy is an emerging cancer therapy with potential great success; however, immune checkpoint inhibitor (e.g., anti-PD-1) has response rates of only 10-30% in solid tumor because of the immunosuppressive tumor microenvironment (TME). This affliction can be solved by vascular normalization and TME reprogramming. METHODS: By using the single-cell RNA sequencing (scRNAseq) approach, we tried to find out the reprogramming mechanism that the Fc-VEGF chimeric antibody drug (Fc-VFD) enhances immune cell infiltration in the TME. RESULTS: In this work, we showed that Fc-VEGF121-VEGF165 (Fc-VEGF chimeric antibody drug, Fc-VFD) arrests excess angiogenesis and tumor growth through vascular normalization using in vitro and in vivo studies. The results confirmed that the treatment of Fc-VFD increases immune cell infiltration including cytotoxic T, NK, and M1-macrophages cells. Indeed, Fc-VFD inhibits Lon-induced M2 macrophages polarization that induces angiogenesis. Furthermore, Fc-VFD inhibits the secretion of VEGF-A, IL-6, TGF-ß, or IL-10 from endothelial, cancer cells, and M2 macrophage, which reprograms immunosuppressive TME. Importantly, Fc-VFD enhances the synergistic effect on the combination immunotherapy with anti-PD-L1 in vivo. CONCLUSIONS: In short, Fc-VFD fusion normalizes intratumor vasculature to reprogram the immunosuppressive TME and enhance cancer immunotherapy.
Assuntos
Antineoplásicos , Neoplasias , Humanos , Microambiente Tumoral , Fator A de Crescimento do Endotélio Vascular , Imunoterapia , Antineoplásicos/farmacologia , Imunossupressores/farmacologiaRESUMO
Due to the high mutation rate of influenza virus and the rapid increase of drug resistance, it is imperative to discover host-targeting antiviral agents with broad-spectrum antiviral activity. Considering the discrepancy between the urgent demand of antiviral drugs during an influenza pandemic and the long-term process of drug discovery and development, it is feasible to explore host-based antiviral agents and strategies from antiviral drugs on the market. In the current study, the antiviral mechanism of arbidol (ARB), a broad-spectrum antiviral drug with potent activity at early stages of viral replication, was investigated from the aspect of hemagglutinin (HA) receptors of host cells. N-glycans that act as the potential binding receptors of HA on 16-human bronchial epithelial (16-HBE) cells were comprehensively profiled for the first time by using an in-depth glycomic approach based on TiO2-PGC chip-Q-TOF MS. Their relative levels upon the treatment of ARB and virus were carefully examined by employing an ultra-high sensitive qualitative method based on Chip LC-QQQ MS, showing that ARB treatment led to significant and extensive decrease of sialic acid (SA)-linked N-glycans (SA receptors), and thereby impaired the virus utilization on SA receptors for rolling and entry. The SA-decreasing effect of ARB was demonstrated to result from its inhibitory effect on sialyltransferases (ST), ST3GAL4 and ST6GAL1 of 16-HBE cells. Silence of STs, natural ST inhibitors, as well as sialidase treatment of 16-HBE cells, resulted in similar potent antiviral activity, whereas ST-inducing agent led to the diminished antiviral effect of ARB. These observations collectively suggesting the involvement of ST inhibition in the antiviral effect of ARB. IMPORTANCE This study revealed, for the first time, that ST inhibition and the resulted destruction of SA receptors of host cells may be an underlying mechanism for the antiviral activity of ARB. ST inhibition has been proposed as a novel host-targeting antiviral approach recently and several compounds are currently under exploration. ARB is the first antiviral drug on the market that was found to possess ST inhibiting function. This will provide crucial evidence for the clinical usages of ARB, such as in combination with neuraminidase (NA) inhibitors to exert optimized antiviral effect, etc. More importantly, as an agent that can inhibit the expression of STs, ARB can serve as a novel lead compound for the discovery and development of host-targeting antiviral drugs.
Assuntos
Indóis , Sialiltransferases , Sulfetos , Antivirais/farmacologia , Antivirais/uso terapêutico , Linhagem Celular , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Células Epiteliais , Glicômica , Hemaglutininas , Humanos , Indóis/farmacologia , Indóis/uso terapêutico , Neuraminidase/farmacologia , Polissacarídeos/metabolismo , Sialiltransferases/antagonistas & inibidores , Sulfetos/farmacologia , Sulfetos/uso terapêuticoRESUMO
Since the pandemic of COVID-19 has intensely struck human society, small animal model for this infectious disease is in urgent need for basic and pharmaceutical research. Although several COVID-19 animal models have been identified, many of them show either minimal or inadequate pathophysiology after SARS-CoV-2 challenge. Here, we describe a new and versatile strategy to rapidly establish a mouse model for emerging infectious diseases in one month by multi-route, multi-serotype transduction with recombinant adeno-associated virus (AAV) vectors expressing viral receptor. In this study, the proposed approach enables profound and enduring systemic expression of SARS-CoV-2-receptor hACE2 in wild-type mice and renders them vulnerable to SARS-CoV-2 infection. Upon virus challenge, generated AAV/hACE2 mice showed pathophysiology closely mimicking the patients with severe COVID-19. The efficacy of a novel therapeutic antibody cocktail RBD-chAbs for COVID-19 was tested and confirmed by using this AAV/hACE2 mouse model, further demonstrating its successful application in drug development.
Assuntos
COVID-19 , Doenças Transmissíveis Emergentes , Modelos Animais de Doenças , Células 3T3 , Enzima de Conversão de Angiotensina 2/genética , Animais , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/uso terapêutico , COVID-19/imunologia , COVID-19/patologia , COVID-19/fisiopatologia , Chlorocebus aethiops , Dependovirus/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Transdução Genética , Células VeroRESUMO
Acquired drug resistance is a major obstacle in cancer therapy. Recent studies revealed that reprogramming of tRNA modifications modulates cancer survival in response to chemotherapy. However, dynamic changes in tRNA modification were not elucidated. In this study, comparative analysis of the human cancer cell lines and their taxol resistant strains based on tRNA mapping was performed by using UHPLC-MS/MS. It was observed for the first time in all three cell lines that 4-demethylwyosine (imG-14) substitutes for hydroxywybutosine (OHyW) due to tRNA-wybutosine synthesizing enzyme-2 (TYW2) downregulation and becomes the predominant modification at the 37th position of tRNAphe in the taxol-resistant strains. Further analysis indicated that the increase in imG-14 levels is caused by downregulation of TYW2. The time courses of the increase in imG-14 and downregulation of TYW2 are consistent with each other as well as consistent with the time course of the development of taxol-resistance. Knockdown of TYW2 in HeLa cells caused both an accumulation of imG-14 and reduction in taxol potency. Taken together, low expression of TYW2 enzyme promotes the cancer survival and resistance to taxol therapy, implying a novel mechanism for taxol resistance. Reduction of imG-14 deposition offers an underlying rationale to overcome taxol resistance in cancer chemotherapy.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Paclitaxel/farmacologia , Processamento Pós-Transcricional do RNA/genética , RNA Neoplásico/química , RNA de Transferência de Fenilalanina/química , Células A549 , Sequência de Bases , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos/fisiologia , Feminino , Regulação Enzimológica da Expressão Gênica , Técnicas de Silenciamento de Genes , Guanosina/análogos & derivados , Guanosina/química , Guanosina/metabolismo , Células HeLa , Humanos , Estrutura Molecular , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Conformação de Ácido Nucleico , Neoplasias Ovarianas/patologia , RNA Neoplásico/fisiologia , RNA de Transferência de Fenilalanina/fisiologia , Espectrometria de Massas em Tandem , Ensaio Tumoral de Célula-TroncoRESUMO
Resistance of cancer cells to chemotherapy is a significant clinical concern and mechanisms regulating cell death in cancer therapy, including apoptosis, autophagy or necrosis, have been extensively investigated over the last decade. Accordingly, the identification of medicinal compounds against chemoresistant cancer cells via new mechanism of action is highly desired. Autophagy is important in inducing cell death or survival in cancer therapy. Recently, novel autophagy activators isolated from natural products were shown to induce autophagic cell death in apoptosis-resistant cancer cells in a calcium-dependent manner. Therefore, enhancement of autophagy may serve as additional therapeutic strategy against these resistant cancers. By computational docking analysis, biochemical assays, and advanced live-cell imaging, we identified that neferine, a natural alkaloid from Nelumbo nucifera, induces autophagy by activating the ryanodine receptor and calcium release. With well-known apoptotic agents, such as staurosporine, taxol, doxorubicin, cisplatin and etoposide, utilized as controls, neferine was shown to induce autophagic cell death in a panel of cancer cells, including apoptosis-defective and -resistant cancer cells or isogenic cancer cells, via calcium mobilization through the activation of ryanodine receptor and Ulk-1-PERK and AMPK-mTOR signaling cascades. Taken together, this study provides insights into the cytotoxic mechanism of neferine-induced autophagy through ryanodine receptor activation in resistant cancers.
Assuntos
Apoptose/efeitos dos fármacos , Morte Celular Autofágica/efeitos dos fármacos , Benzilisoquinolinas/farmacologia , Cálcio/metabolismo , Neoplasias/patologia , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Linhagem Celular Tumoral , Medicamentos de Ervas Chinesas , Humanos , Neoplasias/metabolismoRESUMO
The roles of sphingolipids in polycystic ovary syndrome (PCOS) are still unknown. This study aimed to investigate the sphingolipid characteristics for different types of PCOS using liquid chromatography-mass spectrometry (LC-MS). A total of 107 women with PCOS and 37 healthy women as normal controls were studied. PCOS patients were further classified into non-obesity with insulin resistance (IR) (NOIR), obesity with IR (OIR), and non-obesity and non-IR (NIR) subgroups. A total of 87 serum sphingolipids, including 9 sphingosines, 3 sphinganines, 1 sphingosine-1-phosphate (S1P), 19 ceramides (Cers), 1 ceramide-1-phosphate, 44 sphingomyelins (SMs), 4 hexosylceramides, and 6 lactosylceramides (LacCers) were analyzed using an improved sphingolipidomic approach based on LC-MS. Notable elevations in the levels of S1P, Cer, and SM were observed in PCOS patients when compared with healthy women, and SM species with long saturated acyl chains showed potential as novel biomarkers of PCOS. In addition, the level of LacCer was only elevated in NIR, and there was almost no change in NOIR and OIR. This study is the first to report the comprehensive sphingolipidomic profiling of different subgroups of PCOS with or without IR or obesity and suggests that serum sphingolipids might be useful as diagnostic biomarkers for different types of PCOS.
Assuntos
Síndrome do Ovário Policístico/metabolismo , Esfingolipídeos/metabolismo , Adulto , Estudos de Casos e Controles , Análise Discriminante , Feminino , Humanos , Análise dos Mínimos Quadrados , Lipidômica , Síndrome do Ovário Policístico/sangue , Esfingolipídeos/sangueRESUMO
BACKGROUND: Resistance to chemotherapy drugs (e.g. taxol) has been a major obstacle in successful cancer treatment. In A549 human lung adenocarcinoma, acquired resistance to the first-line chemotherapy taxol has been a critical problem in clinics. Sphingolipid (SPL) controls various aspects of cell growth, survival, adhesion, and motility in cancer, and has been gradually regarded as a key factor in drug resistance. To better understand the taxol-resistant mechanism, a comprehensive sphingolipidomic approach was carried out to investigate the sphingolipid metabolism in taxol-resistant strain of A549 cell (A549T). METHODS: A549 and A549T cells were extracted according to the procedure with optimal condition for SPLs. Sphingolipidomic analysis was carried out by using an UHPLC coupled with quadrupole time-of-flight (Q-TOF) MS system for qualitative profiling and an UHPLC coupled with triple quadrupole (QQQ) MS system for quantitative analysis. The differentially expressed sphingolipids between taxol-sensitive and -resistant cells were explored by using multivariate analysis. RESULTS: Based on accurate mass and characteristic fragment ions, 114 SPLs, including 4 new species, were clearly identified. Under the multiple reaction monitoring (MRM) mode of QQQ MS, 75 SPLs were further quantified in both A549 and A549T. Multivariate analysis explored that the levels of 57 sphingolipids significantly altered in A549T comparing to those of A549 (p < 0.001 and VIP > 1), including 35 sphingomyelins (SMs), 14 ceramides (Cers), 3 hexosylceramides (HexCers), 4 lactosylceramides (LacCers) and 1 sphingosine. A significant decrease of SM and Cer levels and overall increase of HexCer and LacCer represent the major SPL metabolic characteristic in A549T. CONCLUSIONS: This study investigated sphingolipid profiles in human lung adenocarcinoma cell lines, which is the most comprehensive sphingolipidomic analysis of A549 and A549T. To some extent, the mechanism of taxol-resistance could be attributed to the aberrant sphingolipid metabolism, "inhibition of the de novo synthesis pathway" and "activation of glycosphingolipid pathway" may play the dominant role for taxol-resistance in A549T. This study provides insights into the strategy for clinical diagnosis and treatment of taxol resistant lung cancer.
Assuntos
Células A549 , Resistencia a Medicamentos Antineoplásicos , Neoplasias Pulmonares/metabolismo , Paclitaxel/farmacologia , Esfingolipídeos , Células A549/química , Células A549/efeitos dos fármacos , Células A549/metabolismo , Cromatografia Líquida , Biologia Computacional , Humanos , Espectrometria de Massas , Análise de Componente Principal , Esfingolipídeos/análise , Esfingolipídeos/química , Esfingolipídeos/metabolismoRESUMO
In this study, we used Ultra Performance Liquid Chromatography-Time-of-Flight Mass Spectrometry(UPLC-TOF-MS)to identify the chemical constituents in both ethanol and water extract of Polygonum capitatum. A Waters ACQUITY UPLC BEH C18 column(2.1 mm×100 mm,1.7 µm) was used for separation. The mobile phase was consisted of(A) 0.10% formic acid in water and(B)0.10% formic acid in acetonitrile, and the flow rate was 0.35 mLâ¢min⻹. ESI source in negative ion mode was used for MS detection. Structural identification was carried out according to the accurate mass and matching with database. The results showed that flavonoids, polyphenols and lignans were the main components in both extracts. However, the chemical compositions of both extracts were different, e.g. there are less hydrolyzable tannins, loss of ellagic acid and more anthocyanins in ethanol extract. In a conclusion, this study provides an important scientific basis for identifying the active ingredients in P. capitatum, which also help to reveal the pharmacological effect of P. capitatum.
Assuntos
Medicamentos de Ervas Chinesas/análise , Extratos Vegetais/análise , Polygonum/química , Cromatografia Líquida de Alta Pressão , Etanol , Flavonoides/análise , Lignanas/análise , Polifenóis/análise , Espectrometria de Massas em Tandem , ÁguaRESUMO
Cancers illustrating resistance towards apoptosis is one of the main factors causing clinical failure of conventional chemotherapy. Innovative therapeutic methods which can overcome the non-apoptotic phenotype are needed. The AMP-activated protein kinase (AMPK) is the central regulator of cellular energy homeostasis, metabolism, and autophagy. Our previous study showed that the identified natural AMPK activator is able to overcome apoptosis-resistant cancer via autophagic cell death. Therefore, AMPK is an ideal pharmaceutical target for chemoresistant cancers. Here, we unravelled that the bisbenzylisoquinoline alkaloid thalidezine is a novel direct AMPK activator by using biolayer interferometry analysis and AMPK kinase assays. The quantification of autophagic EGFP-LC3 puncta demonstrated that thalidezine increased autophagic flux in HeLa cancer cells. In addition, metabolic stress assay confirmed that thalidezine altered the energy status of our cellular model. Remarkably, thalidezine-induced autophagic cell death in HeLa or apoptosis-resistant DLD-1 BAX-BAK DKO cancer cells was abolished by addition of autophagy inhibitor (3-MA) and AMPK inhibitor (compound C). The mechanistic role of autophagic cell death in resistant cancer cells was further supported through the genetic removal of autophagic gene7 (Atg7). Overall, thalidezine is a novel AMPK activator which has great potential to be further developed into a safe and effective intervention for apoptosis- or multidrug-resistant cancers.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Linhagem Celular Tumoral , HumanosRESUMO
Drug resistance elicited by cancer cells continue to cause huge problems world-wide, for example, tens of thousands of patients are suffering from taxol-resistant human ovarian cancer. However, its biochemical mechanisms remain unclear. Sphingolipid metabolic dysregulation has been increasingly regarded as one of the drug-resistant mechanisms for various cancers, which in turn provides potential targets for overcoming the resistance. In the current study, a well-established LC-MS based sphingolipidomic approach was applied to investigate the sphingolipid metabolism of A2780 and taxol-resistant A2780 (A2780T) human ovarian cancer cell lines. 102 sphingolipids (SPLs) were identified based on accurate mass and characteristic fragment ions, among which 12 species have not been reported previously. 89 were further quantitatively analyzed by using multiple reaction monitoring technique. Multivariate analysis revealed that the levels of 52 sphingolipids significantly altered in A2780T cells comparing to those of A2780 cells. These alterations revealed an overall increase of sphingomyelin levels and significant decrease of ceramides, hexosylceramides and lactosylceramides, which concomitantly indicated a deviated SPL metabolism in A2780T. This is the most comprehensive sphingolipidomic analysis of A2780 and A2780T, which investigated significantly changed sphingolipid profile in taxol-resistant cancer cells. The aberrant sphingolipid metabolism in A2780T could be one of the mechanisms of taxol-resistance.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Neoplasias Ovarianas/metabolismo , Esfingolipídeos/análise , Linhagem Celular Tumoral , Cromatografia Líquida/métodos , Feminino , Humanos , Paclitaxel/farmacologia , Espectrometria de Massas em Tandem/métodosRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Toad venom and toad skin have been widely used for treating various cancers in China. Bufadienolides are regarded as the main anticancer components of toad venom, but the difference on composition and anticancer activities of bufadienolides between toad venom and toad skin remains unclear. METHODS: Fractions enriched with free and conjugated bufadienolides were prepared from toad venom and toad skin. Bufadienolides in each fraction were comprehensively profiled by using a versatile UHPLC-TOF-MS method. Relative contents of major bufadienolides were determined by using three bufogenins and one bufotoxin as marker compounds with validated UHPLC-TOF-MS method. Furthermore, cytotoxicity of the fractions was examined by MTT assay. RESULTS: Two fractions, i.e., bufogenin and bufotoxin fractions (TV-F and TV-C) were isolated from toad venom, and one bufotoxin fraction (TS-C) was isolated from toad skin. Totally 56 bufadienolides in these three fractions were identified, and 29 were quantified or semi-quantified. Bufotoxins were identified in both toad venom and toad skin, whereas bufogenins exist only in toad venom. Bufalin-3-conjugated bufotoxins are major components in toad venom, whereas cinobufotalin and cinobufagin-3-conjugated bufotoxins are main bufotoxins in toad skin. MTT assay revealed potent cytotoxicity of all the fractions in an order of TV-F>TV-C>TS-C. CONCLUSIONS: Our study represents the most comprehensive investigation on the chemical profiles of toad venom and toad skin from both qualitative and quantitative aspects. Eight bufotoxins were identified in toad skin responsible for the cytotoxicity for the first time. Our research provides valuable chemical evidence for the appropriate processing method, quality control and rational exploration of toad skin and toad venom for the development of anticancer medicines.
Assuntos
Venenos de Anfíbios/química , Antineoplásicos/isolamento & purificação , Bufanolídeos/isolamento & purificação , Pele/química , Animais , Antineoplásicos/farmacologia , Bufanolídeos/farmacologia , Bufo bufo , Células CACO-2 , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Células Hep G2 , Humanos , Células MCF-7 , Espectrometria de MassasRESUMO
Drug resistance hinder most cancer chemotherapies and leads to disease recurrence and poor survival of patients. Resistance of cancer cells towards apoptosis is the major cause of these symptomatic behaviours. Here, we showed that isoquinoline alkaloids, including liensinine, isoliensinine, dauricine, cepharanthine and hernandezine, putatively induce cytotoxicity against a repertoire of cancer cell lines (HeLa, A549, MCF-7, PC3, HepG2, Hep3B and H1299). Proven by the use of apoptosis-resistant cellular models and autophagic assays, such isoquinoline alkaloid-induced cytotoxic effect involves energy- and autophagy-related gene 7 (Atg7)-dependent autophagy that resulted from direct activation of AMP activated protein kinase (AMPK). Hernandezine possess the highest efficacy in provoking such cell death when compared with other examined compounds. We confirmed that isoquinoline alkaloid is structurally varied from the existing direct AMPK activators. In conclusion, isoquinoline alkaloid is a new class of compound that induce autophagic cell death in drug-resistant fibroblasts or cancers by exhibiting its direct activation on AMPK.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Autofagia/efeitos dos fármacos , Benzilisoquinolinas/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Western Blotting , Proliferação de Células/efeitos dos fármacos , Medicamentos de Ervas Chinesas/química , Embrião de Mamíferos/citologia , Embrião de Mamíferos/efeitos dos fármacos , Embrião de Mamíferos/enzimologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Citometria de Fluxo , Humanos , Técnicas Imunoenzimáticas , Camundongos , Microscopia de Fluorescência , Neoplasias/enzimologia , Células Tumorais CultivadasRESUMO
Mutant huntingtin aggregation is highly associated with the pathogenesis of Huntington's disease, an adult-onset autosomal dominant disorder, which leads to a loss of motor control and decline in cognitive function. Recent literature has revealed the protective role of autophagy in neurodegenerative diseases through degradation of mutant toxic proteins, including huntingtin or a-synuclein. Through the GFP-LC3 autophagy detection platform, we have identified neferine, isolated from the lotus seed embryo of Nelumbo nucifera, which is able to induce autophagy through an AMPK-mTOR-dependent pathway. Furthermore, by overexpressing huntingtin with 74 CAG repeats (EGFP-HTT 74) in PC-12 cells, neferine reduces both the protein level and toxicity of mutant huntingtin through an autophagy-related gene 7 (Atg7)-dependent mechanism. With the variety of novel active compounds present in medicinal herbs, our current study suggests the possible protective mechanism of an autophagy inducer isolated from Chinese herbal medicine, which is crucial for its further development into a potential therapeutic agent for neurodegenerative disorders in the future.
Assuntos
Autofagia/efeitos dos fármacos , Benzilisoquinolinas/farmacologia , Medicamentos de Ervas Chinesas/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Doença de Huntington/patologia , Proteínas Mutantes/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Adulto , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Proliferação de Células/efeitos dos fármacos , Medicamentos de Ervas Chinesas/química , Citometria de Fluxo , Humanos , Proteína Huntingtina , Doença de Huntington/tratamento farmacológico , Doença de Huntington/genética , Proteínas Mutantes/genética , Proteínas do Tecido Nervoso/genética , Células PC12 , RNA Mensageiro/genética , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
Hydrogen peroxide (H2O2), normally used in the prevention of local bacterial infection, is also used for clinical debridement in bone and joint surgery. Studies show that treatment with H2O2 can cause cartilage damage, leading to postoperative complications. H2O2 can induce apoptosis because of its strong oxidation activity. To investigate the molecular mechanisms of H2O2-induced chondrocyte apoptosis, the rat chondrocytes from the knee joint were used as a cell model for this study. The results showed that the H2O2-treated cells survived at a decreased rate, with apoptosis in a great number of chondrocytes. The expression of pro-apoptotic factors of chondrocytes, Bcl-2 and Bcl-xl, were downregulated, while the pro-apoptotic factor Bax upregulated. Two important factors ERK and p38 in MAPK signaling pathway were phosphorylated at a higher level, leading to apoptosis of chondrocytes. This study described the molecular mechanism of H2O2-induced chondrocyte apoptosis, providing a theoretical basis for the rational and clinical use of H2O2 as a disinfectant.
Assuntos
Apoptose , Cartilagem/metabolismo , Condrócitos/metabolismo , Desinfetantes/toxicidade , Peróxido de Hidrogênio/toxicidade , Animais , Cartilagem/citologia , Cartilagem/efeitos dos fármacos , Células Cultivadas , Condrócitos/efeitos dos fármacos , Desinfetantes/efeitos adversos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Peróxido de Hidrogênio/efeitos adversos , Sistema de Sinalização das MAP Quinases , Ratos , Ratos Sprague-Dawley , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Resistance of cancer cells to chemotherapy is a significant problem in oncology, and the development of sensitising agents or small-molecules with new mechanisms of action to kill these cells is needed. Autophagy is a cellular process responsible for the turnover of misfolded proteins or damaged organelles, and it also recycles nutrients to maintain energy levels for cell survival. In some apoptosis-resistant cancer cells, autophagy can also enhance the efficacy of anti-cancer drugs through autophagy-mediated mechanisms of cell death. Because the modulation of autophagic processes can be therapeutically useful to circumvent chemoresistance and enhance the effects of cancer treatment, the identification of novel autophagic enhancers for use in oncology is highly desirable. Many novel anti-cancer compounds have been isolated from natural products; therefore, we worked to discover natural, anti-cancer small-molecule enhancers of autophagy. Here, we have identified a group of natural alkaloid small-molecules that function as novel autophagic enhancers. These alkaloids, including liensinine, isoliensinine, dauricine and cepharanthine, stimulated AMPK-mTOR dependent induction of autophagy and autophagic cell death in a panel of apoptosis-resistant cells. Taken together, our work provides novel insights into the biological functions, mechanisms and potential therapeutic values of alkaloids for the induction of autophagy.
Assuntos
Alcaloides/química , Alcaloides/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Autofagia/efeitos dos fármacos , Autofagia/fisiologia , Células HeLa , Humanos , Peso MolecularRESUMO
The emerging field of sphingolipidomics calls for accurate quantitative analyses of sphingolipidome. Existing analytical methods for sphingolipid (SPL) profiling often suffer from isotopic/isomeric interference, leading to the low-abundance, but biologically important SPLs being undetected. In the current study, we have developed an improved sphingolipidomic approach for reliable and sensitive quantification of up to 10 subclasses of cellular SPLs. By integratively utilizing high efficiency chromatographic separation, quadrupole time-of-flight (Q-TOF) and triple quadrupole (QQQ) mass spectrometry (MS), our approach facilitated unambiguous identification of several groups of potentially important but low-abundance SPLs that are usually masked by isotopic/isomeric species and hence largely overlooked in many published methods. The methodology, which featured a modified sample preparation and optimized MS parameters, permitted the measurement of 86 individual SPLs in PC12 cells in a single run, demonstrating great potential for high throughput analysis. The improved characterization, along with increased sensitivity for low-abundance SPL species, resulted in the highest number of SPLs being quantified in a single run in PC12 cells. The improved method was fully validated and applied to a lipidomic study of PC12 cell samples with or without amyloid ß peptide (Aß) treatment, which presents a most precise and genuine sphingolipidomic profile of the PC12 cell line. The adoption of the metabolomics protocol, as described in this study, could avoid misidentification and bias in the measurement of the analytically challenging low-abundance endogenous SPLs, hence achieving informative and reliable sphingolipidomics data relevant to discovery of potential SPL biomarkers for Aß-induced neurotoxicity and neurodegenerative disease.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas/métodos , Neurônios/efeitos dos fármacos , Esfingolipídeos/química , Animais , Limite de Detecção , Células PC12 , Ratos , Testes de ToxicidadeRESUMO
Catechins and procyanidins, together with flavonoid glycosides and terpene trilactones, are three important categories of components in the standard extract of Ginkgo biloba leaves (EGb761). In this research, catechins and proanthocyanidins were found to exist in both the extract of Ginkgo leaves and Ginkgo products. By comparing with reference compounds, six of them were identified as (+)-catechin, (-)-epicatechin, (-)-gallocatechin, (-)-epigallocatechin and procyanidins B1 and B3. The activities of these polyphenols in the inhibition of Aß42 aggregation and the destabilization of preformed fibrils were evaluated using biochemical assays, which showed that all six of the polyphenols, as well as a fraction of the extract of Ginkgo biloba leaves (EGb) containing catechins and procyanidins, exerted potent inhibitory activities towards Aß42 aggregation and could also destabilize the performed fibrils. Catechins and procyanidins can therefore be regarded as the potent active constituents of EGb761 in terms of their inhibition of Aß42 aggregation and destabilization of the fibrils. Although quantitative mass spectroscopic analysis revealed that the catechins and procyanidins are only present in low concentrations in EGb761, these components should be studied in greater detail because of their potent inhibitory effects towards Aß42 aggregation and their ability to destabilize preformed fibrils, especially during the quality control of Ginkgo leaves and the manufacture of Ginkgo products.
Assuntos
Peptídeos beta-Amiloides/antagonistas & inibidores , Benzopiranos/química , Ginkgo biloba/química , Fragmentos de Peptídeos/antagonistas & inibidores , Extratos Vegetais/química , Folhas de Planta/química , Peptídeos beta-Amiloides/química , Benzopiranos/isolamento & purificação , Biflavonoides/química , Biflavonoides/isolamento & purificação , Catequina/análogos & derivados , Catequina/química , Catequina/isolamento & purificação , Humanos , Fragmentos de Peptídeos/química , Proantocianidinas/química , Proantocianidinas/isolamento & purificação , Relação Estrutura-AtividadeRESUMO
Multicomponent metabolic profile of notoginseng saponins in artificial gastric juice was qualitatively and quantitatively investigated, showing that ginsenosides were transformed via multiple pathways including deglycosylation, dehydration, hydration, and oxygenation. A total of 83 metabolites was identified by using UPLC-Q-TOF-MS, among which 16 new dammarane glycosides were further characterized by comparing with synthesized authentic compounds. Transformation time-course of notoginseng saponins in artificial gastric juice was quantitatively measured for the first time, showing rapid degradation of primary ginsenosides and concomitant formation of deglycosylation, hydration, and dehydration products. It was further demonstrated that the resultant metabolites exhibited enhanced cytotoxicity toward cancer cells. The extensive metabolism of ginsenosides within a transit time span in stomach, together with the formation of metabolites with diversified chemical structures possessing enhanced biological activities, indicated an important role of transformation in gastric juice in the systematic effects of ginsenosides.
Assuntos
Suco Gástrico/metabolismo , Ginsenosídeos/metabolismo , Ginsenosídeos/farmacologia , Panax notoginseng/metabolismo , Extratos Vegetais/metabolismo , Extratos Vegetais/farmacologia , Biotransformação , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Mucosa Gástrica/metabolismo , Ginsenosídeos/química , Humanos , Espectrometria de Massas , Modelos Biológicos , Estrutura Molecular , Panax notoginseng/química , Extratos Vegetais/químicaRESUMO
The method of 2dimensional gel electrophoresis (2-DE) has been widely used for the proteomic profiling of solid biological samples, however, the analytical conditions have not been optimized. The present study optimized the major conditions of 2DE for determining the protein contents of solid tumor tissues, through enhancement of the separation efficiency and resolution. Three major analytical conditions of 2DE analysis, namely protein extraction, focusing time for isoelectric focusing (IEF), and prereduction and alkylation prior to IEF, were carefully examined so that the optimal parameters and procedures were achieved. The use of a bead mill for protein extraction resulted in a higher protein yield in a minimal processing time. An optimal focusing time for IEF was established which improved the 2DE image quality and reproducibility. Furthermore, reduction and alkylation of the protein sample prior to IEF reduced the horizontal streaking caused by oxidation and improved the resolution at the cathode. The optimized 2DE analysis enabled the detection of 20% more protein spots compared with the previous reported conditions, with higher image quality and reproducibility. Accordingly, the optimized conditions may be used in the 2DE analysis of tumor tissue samples, by which novel biomarkers of cancerous diseases and molecular targets of drugs are expected to be identified.
Assuntos
Eletroforese em Gel Bidimensional/métodos , Neoplasias/genética , Proteômica/métodos , Regulação Neoplásica da Expressão Gênica , Humanos , Focalização Isoelétrica/métodos , Terapia de Alvo Molecular , Neoplasias/patologia , Biossíntese de Proteínas/genéticaRESUMO
Peptide isoelectric focusing (IEF) is a common technique used in two-dimensional liquid chromatography tandem mass spectrometry (2D-LC-MS/MS) proteomic workflow, in which the tryptic peptide is first pre-fractionated based on pI values before being subjected to reverse phase LC-MS analysis. Although this method has been widely used by many research groups, a systemic study on the optimal conditions and fundamental parameters influencing the experimental outcomes has been lacking, including the effect of peptide extraction methods, the extent of pre-fractionation, and the choice of pH range. In this study, we compared the effect of different parameters on the numbers of peptides and proteins identified using two complex mouse proteomes. The results indicated that extraction of peptides from immobilized pH gradient (IPG) strips by sequential elution of increasingly organic solvents provided the highest number of peptide identification. In addition, we showed that approximately 45 more unique proteins were identified for every additional fraction collected during peptide IEF. Although narrow pH ranges provided higher resolution in peptide separation as expected, different pH ranges yielded similar numbers of peptide and protein identification. Overall, we demonstrated that the extraction solvent influenced the numbers of peptide and protein identification and quantitatively demonstrated the advantage of extensive fractionation and the performance of different pH ranges in practice.