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Pro-survival BCL-2 proteins prevent the initiation of intrinsic apoptosis (mitochondria-dependent pathway) by inhibiting the pro-apoptotic proteins BAX and BAK, while BH3-only proteins promote apoptosis by blocking pro-survival BCL-2 proteins. Disruptions in this delicate balance contribute to cancer cell survival and chemoresistance. Recent advances in cancer therapeutics involve a new generation of drugs known as BH3-mimetics, which are small molecules designed to mimic the action of BH3-only proteins. Promising effects have been observed in patients with hematological and solid tumors undergoing treatment with these agents. However, the rapid emergence of mitochondria-dependent resistance to BH3-mimetics has been reported. This resistance involves increased mitochondrial respiration, altered mitophagy, and mitochondria with higher and tighter cristae. Conversely, mutations in isocitrate dehydrogenase 1 and 2, catalyzing R-2-hydroxyglutarate production, promote sensitivity to venetoclax. This evidence underscores the urgency for comprehensive studies on bioenergetics-based adaptive responses in both BH3 mimetics-sensitive and -resistant cancer cells. Ongoing clinical trials are evaluating BH3-mimetics in combination with standard chemotherapeutics. In this article, we discuss the role of mitochondrial bioenergetics in response to BH3-mimetics and explore potential therapeutic opportunities through metabolism-targeting strategies.
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Antineoplásicos , Metabolismo Energético , Mitocondrias , Neoplasias , Proteínas Proto-Oncogénicas c-bcl-2 , Humanos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Neoplasias/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Metabolismo Energético/efectos de los fármacos , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , AnimalesRESUMEN
Lung cancer is one of the most commonly occurring cancer types that accounts for almost 2 million cases per year. Its resistance to anticancer drugs, failure of new molecules in clinical trials, severe side-effects of current treatments, and its recurrence limit the success of anticancer therapies. Nanotherapeutic agents offer several advantages over conventional anticancer therapies, including improved retention in tumors, specificity, and anticancer effects at lower concentrations, hence reducing the side-effects. Here, we have explored the anticancer activity of silver nanoparticles synthesized in Viridibacillus sp. enriched culture medium for the first time. Such green nanoparticles, synthesized by biological systems, are superior to chemically synthesized ones in terms of their environmental footprint and production cost, and have one crucial advantage of excellent stability owing to their biological corona. To assess anticancer activity of these nanoparticles, we used conventional 2D cultured A549 cells as well as 3D spheroids of A549 cells. In both models of lung cancer, our silver nanoparticles diminished cell proliferation, arrested DNA synthesis, and showed a dose dependent cytotoxic effect. The nanoparticles damaged the DNA and mitochondrial structures in both A549 cells and A549 spheroids, leading to mitochondrial depolarization and increased cell permeability. Low lethal median doses (LD50) for 2D cultured A549 cells (1 µg/ml) and for A549 spheroids (13 µg/ml) suggest that our nanoparticles are potent anticancer agents. We also developed in vitro tumor progression model and in vitro tumor size model using 3D spheroids to test anticancer potential of our nanoparticles which otherwise would require longer experimental duration along with large number of animals and trained personnel. In these models, our nanoparticles showed strong dose dependent anticancer activity. In case of in vitro tumor progression model, the A549 cells failed to form tight spheroidal mass and showed increased dead cell fraction since day 1 as compared to control. On the other hand, in case of in vitro tumor size model, the 4 and 8 µg/ml nanoparticle treatment led to reduction in spheroid size from 615 ± 53 µm to 440 ± 45 µm and 612 ± 44 µm to 368 ± 62 µm respectively, within the time span of 3 days post treatment. We believe that use of such novel experimental models offers excellent and fast alternative to in vivo studies, and to the best of our knowledge, this is the first report that gives proof-of-concept for use of such novel in vitro cancer models to test anticancer agents such as Viridibacilli culture derived silver nanoparticles. Based on our results, we propose that these nanoparticles offer an interesting alternative for anticancer therapies, especially if they can be combined with classical anticancer drugs.
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Glioblastoma multiforme (GBM) is one of the deadliest cancers. Temozolomide (TMZ) is the most common chemotherapy used for GBM patients. Recently, combination chemotherapy strategies have had more effective antitumor effects and focus on slowing down the development of chemotherapy resistance. A combination of TMZ and cholesterol-lowering medications (statins) is currently under investigation in in vivo and clinical trials. In our current investigation, we have used a triple-combination therapy of TMZ, Simvastatin (Simva), and acetylshikonin, and investigated its apoptotic mechanism in GBM cell lines (U87 and U251). We used viability, apoptosis, reactive oxygen species, mitochondrial membrane potential (MMP), caspase-3/-7, acridine orange (AO) and immunoblotting autophagy assays. Our results showed that a TMZ/Simva/ASH combination therapy induced significantly more apoptosis compared to TMZ, Simva, ASH, and TMZ/Simva treatments in GBM cells. Apoptosis via TMZ/Simva/ASH treatment induced mitochondrial damage (increase of ROS, decrease of MMP) and caspase-3/7 activation in both GBM cell lines. Compared to all single treatments and the TMZ/Simva treatment, TMZ/Simva/ASH significantly increased positive acidic vacuole organelles. We further confirmed that the increase of AVOs during the TMZ/Simva/ASH treatment was due to the partial inhibition of autophagy flux (accumulation of LC3ß-II and a decrease in p62 degradation) in GBM cells. Our investigation also showed that TMZ/Simva/ASH-induced cell death was depended on autophagy flux, as further inhibition of autophagy flux increased TMZ/Simva/ASH-induced cell death in GBM cells. Finally, our results showed that TMZ/Simva/ASH treatment potentially depends on an increase of Bax expression in GBM cells. Our current investigation might open new avenues for a more effective treatment of GBM, but further investigations are required for a better identification of the mechanisms.
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OBJECTIVE: To investigate the role of tumor necrosis factor receptor-associated factor 6 (TRAF6) in regulating Bacillus Calmette-Guérin (BCG)-induced macrophage apoptosis. METHODS: The expression of TRAF6 in peripheral blood samples of 50 patients with active tuberculosis (TB) and 50 healthy individuals were detected using quantitative real-time PCR (qPCR). RAW264.7 macrophages were infected with BCG at different MOI and for different lengths of time, and the changes in expressions of Caspase 3 and TRAF6 were detected with Western blotting and qPCR. In a RAW264.7 cell model of BCG infection with TRAF6 knockdown established using RNA interference technique, the bacterial load was measured and cell apoptotic rate and mitochondrial membrane potential (MMP) were determined with flow cytometry. The expression levels of TRAF6, Caspase 3, PARP, BAX and Bcl-2 in the cells were detected using Western blotting, and the expressions of TRAF6 and Caspase 3 were also examined with immunofluorescence assay. RESULTS: The expression of TRAF6 was significantly upregulated in the peripheral blood of patients with active TB as compared with healthy subjects (P < 0.001). In RAW264.7 cells, BCG infection significantly increased the expressions of Caspase 3 and TRAF6, which were the highest in cells infected for 18 h and at the MOI of 15. TRAF6 knockdown caused a significant increase of bacterial load in BCG-infected macrophages (P=0.05), lowered the cell apoptotic rate (P < 0.001) and reduced the expressions of Caspase 3 (P=0.002) and PARP (P < 0.001). BCG-infected RAW264.7 cells showed a significantly increased MMP (P < 0.001), which was lowered by TRAF6 knockdown (P < 0.001); the cells with both TRAF6 knockdown and BCG infection showed a lowered BAX expression (P=0.005) and an increased expression of Bcl-2 (P=0.04). CONCLUSION: TRAF6 promotes BCG-induced macrophage apoptosis by regulating the intrinsic apoptosis pathway.
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Mycobacterium bovis , Factor 6 Asociado a Receptor de TNF , Apoptosis , Vacuna BCG , Caspasa 3/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Macrófagos , Mycobacterium bovis/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Factor 6 Asociado a Receptor de TNF/metabolismo , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Isatis tinctoria and its indigo dyes have already provided highly active anti-leukaemic lead compounds, with the focus mainly being on indirubin, whereas indigo itself is inactive. There are many more indigoids to find in this plant extract, for example, quingdainone, an indigoid derived from tryptanthrin. We present here a new synthesis of hitherto neglected substituted quingdainones, which is very necessary due to their poor solubility behaviour, and a structure-dependent anti-leukaemic activity study of a number of compounds. Substituted α-phenylaminoacrylic acid was synthesised by hydrogen sulfide extrusion from an analogue mercaptoacetic acid, available from the condensation of rhodanin and a substituted tryptanthrin. It is shown that just improving water solubility does not increase anti-leukaemic activity, since a quingdainone carboxylic acid is inactive compared to dihydroxyquingdainone. The most effective compound, dihydroxyquingdainone with an AC50 of 7.5 µmole, is further characterised, revealing its ability to overcome multidrug resistance in leukaemia cells (Nalm-6/BeKa) with p-glycoprotein expression.
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Citostáticos , Leucemia , Linfoma , Apoptosis , Caspasa 3 , Carmin de Índigo , Leucemia/tratamiento farmacológico , Hojas de la PlantaRESUMEN
Fish gills are in direct contact with the surrounding pollutants, and thus, potentially more vulnerable to microplastics (MPs) and heavy metals. The present study aimed to evaluate the long-term exposure effects of MPs and copper (Cu) in the gills of adult zebrafish (Danio rerio). To this end, zebrafish were exposed to MPs (2 mg/L), Cu (Cu25, 25 µg/L) and their mixture (Cu25 + MPs) for 30 days, and then oxidative stress, detoxification, antioxidant, metabolic and neurotoxicity enzymes/genes, as well serotonergic system and apoptosis genes, were evaluated in gills. In the mixture group, ROS levels were increased, while CAT and GPx activities were inhibited, indicating the induction of oxidative stress in zebrafish gills. This was followed by an increase of LPO levels and potential oxidative damage in zebrafish gills. The tryptophan hydroxylase 1a (tph1a) and caspase-3 (casp3) genes were significantly upregulated in Cu25 + MPs group, indicating a potential dysregulation of serotonin synthesis and apoptosis pathways, respectively. Overall, the present study contributes to improving the knowledge about the response of aquatic organisms to MPs and the potential ecological risk that these particles represent to the ecosystems.
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Microplásticos , Contaminantes Químicos del Agua , Animales , Apoptosis , Cobre/metabolismo , Ecosistema , Branquias/metabolismo , Microplásticos/toxicidad , Estrés Oxidativo , Plásticos/toxicidad , Contaminantes Químicos del Agua/metabolismo , Pez Cebra/metabolismoRESUMEN
PURPOSE OF REVIEW: Apoptosis is a major mechanism of cancer cell death. Thus, evasion of apoptosis results in therapy resistance. Here, we review apoptosis modulators in cancer and their recent developments, including MDM2 inhibitors and kinase inhibitors that can induce effective apoptosis. RECENT FINDINGS: Both extrinsic pathways (external stimuli through cell surface death receptor) and intrinsic pathways (mitochondrial-mediated regulation upon genotoxic stress) regulate the complex process of apoptosis through orchestration of various proteins such as members of the BCL-2 family. Dysregulation within these complex steps can result in evasion of apoptosis. However, via the combined evolution of medicinal chemistry and molecular biology, omics assays have led to innovative inducers of apoptosis and inhibitors of anti-apoptotic regulators. Many of these agents are now being tested in cancer patients in early-phase trials. We believe that despite a sluggish speed of development, apoptosis targeting holds promise as a relevant strategy in cancer therapeutics.
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Antineoplásicos , Neoplasias , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Apoptosis , Humanos , Mitocondrias/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2RESUMEN
The black pepper, most commonly used in Indian cuisines for ages, is considered as "king of spices." The present study evaluates the anticancer potential of black pepper and its main constituent, i.e. alkaloid piperine, against human leukemia cell line, K-562 cells. Gas chromatography-mass spectrometry (GC-MS) analysis confirmed the presence of piperine in black pepper extract. The methanolic extract of black pepper (BP-M) and pure piperine (PIP) showed a strong cytotoxic effect against this cell line. Both BP-M and PIP generated apoptotic bodies in K-562 cells and caused nuclear condensation as visualized by fluorescent microscopy, which was further confirmed by flow cytometry analysis. BP-M and PIP also generated reactive oxygen species in K-562 cells as established by flow cytometry. The translation of Bax, caspase-3 and caspase-9 genes was found to be upregulated with subsequent downregulation of Bcl-2 gene. The anti-proliferative effect of both BP-M and PIP was also observed by trypan blue staining and was further confirmed by the downregulated expression of proliferating cell nuclear antigen (PCNA). The molecular docking studies showed the binding of PIP with PCNA and Bcl-2 and supported the in vitro findings. The docking studies also proposed the binding of PIP to ADP binding pocket of Apaf-1 protein. Taken together, these findings signify the anticancer potential of both black pepper and PIP, thus proposing black pepper as a potent nutraceutical for preventing the progression of chronic myeloid leukemia.
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AIM: To investigate the anti-proliferation and apoptosis-inducing effects of sodium aescinate (SA) on retinoblastoma Y79 cells and its mechanism. METHODS: Y79 cells were cultured at different drug concentrations for different periods of time (24, 48, and 72h). The inhibitory effect of SA on proliferation of Y79 cells was detected by the cell counting kit-8 (CCK-8) assay, and the morphology of Y79 cells in each group was observed under an inverted microscope. An IC50 of 48h was selected for subsequent experiments. After pretreatment with SA for 24 and 48h, cellular DNA distribution and apoptosis were detected by flow cytometry. Real-time qunatitative polymerase chain reaction (RT-qPCR) and Western blot were used to assess changes in related genes (CDK1, CyclinB1, Bax, Bcl-2, caspase-9, caspase-8, and caspase-3). RESULTS: SA inhibited proliferation and induced apoptosis of Y79 cells in a time-dependent and concentration-dependent manner. Following its intervention in the cell cycle pathway, SA can inhibit the expression of CDK1 and CyclinB1 at the mRNA and protein levels, and block cells in the G2/M phase. In caspase-related apoptotic pathways, up-regulation of Bax and down-regulation of Bcl-2 caused caspase-9 to self-cleave and further activate caspase-3. What's more, the caspase-8-mediated extrinsic apoptosis pathway was activated, and the activated caspase-8 was released into the cytoplasm to activate caspase-3, which as a member of the downstream apoptotic effect group, initiates a caspase-cascade reaction that induces cell apoptosis. CONCLUSION: SA inhibits the proliferation of Y79 cells by arresting the cell cycle at the G2/M phase, and induces apoptosis via the caspase-related apoptosis pathway, indicating that SA may have promising potential as a chemotherapeutic drug.
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Recently, the incidence of thyroid cancer is increasing worldwide. Papillary thyroid cancer (PTC) is the most common histological type of thyroid cancer. Whole-transcriptome sequence analysis was performed to further understand the primary molecular mechanisms of the occurrence and progression of PTC. Results showed that Eva-1 homolog A (EVA1A) may be a potential gene for the PTC-associated gene in thyroid cancer. In this work, the role of EVA1A expression in thyroid cancer was investigated. Real-time PCR was performed to detect the expression level of EVA1A in 43 pairs of PTC and four thyroid cancer cell lines. The Cancer Genome Atlas (TCGA) database was used to evaluate the relationship between the expression level of EVA1A and the pathological feature of PTC. The logistic regression analysis of the TCGA data set indicated that the expression of EVA1A was an independent risk factor for tumour, nde and metastasis (TNM) in PTC. This study shows the down-regulation of EVA1A inhibited the colony formation, proliferation, migration and invasion of PTC cell lines. In the protein level, knockdown of EVA1A can regulate the expression of N-cadherin, vimentin, Bcl-xL, Bax, YAP and TAZ. This study indicated that EVA1A was an oncogene associated with PTC.
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Proteínas de la Membrana/metabolismo , Proteínas Serina-Treonina Quinasas , Transducción de Señal , Cáncer Papilar Tiroideo/metabolismo , Neoplasias de la Tiroides/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adulto , Anciano , Antígenos CD/metabolismo , Apoptosis , Cadherinas/metabolismo , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Transición Epitelial-Mesenquimal/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Vía de Señalización Hippo , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Persona de Mediana Edad , Invasividad Neoplásica , Proteínas Serina-Treonina Quinasas/metabolismo , Factores de Riesgo , Programas Informáticos , Cáncer Papilar Tiroideo/patología , Neoplasias de la Tiroides/patología , Factores de Transcripción/metabolismo , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ , Vimentina/metabolismo , Proteínas Señalizadoras YAP , Proteína X Asociada a bcl-2/metabolismo , Proteína bcl-X/metabolismoRESUMEN
Podocyte injury leads to impaired filtration barrier function of the kidney that underlies the pathophysiology of idiopathic nephrotic syndrome (INS), the most common NS occurring in children. The heat shock protein 90 (Hsp90) is involved in the regulation of apoptosis in a variety of cell types, however, little is known about its role in podocytes and whether it associated with NS. Here, we show that Hsp90 is upregulated in glomeruli podocytes from mice with adriamycin (ADR)-induced nephropathy, and that it is also upregulated in an immortalized podocyte cell line treated with ADR in vitro, together suggesting an association of Hsp90 upregulation in podocytes with NS pathogenesis. Functionally, Hsp90 inhibition with PU-H71 aggravates ADR-induced podocyte apoptosis and worsens the impairment of filtration barrier function. Mechanistically, Hsp90 inhibition with PU-H71 enhances the activation of intrinsic apoptotic pathway, and moreover, blockade of podocyte apoptosis with zVAD-fmk (aVAD), a pan-caspase inhibitor, abrogates effects of Hsp90 inhibition on filtration barrier function of ADR-treated podocytes, thus demonstrating that Hsp90 inhibition aggravates ADR-induced podocyte injury through intrinsic apoptosis pathway. In sum, this study reveals a detrimental role of Hsp90 inhibition in podocyte injury, which may offer it as a potential therapeutic target in NS therapy.
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Antibióticos Antineoplásicos/administración & dosificación , Apoptosis/efectos de los fármacos , Doxorrubicina/administración & dosificación , Proteínas HSP90 de Choque Térmico/genética , Síndrome Nefrótico/genética , Podocitos/efectos de los fármacos , Clorometilcetonas de Aminoácidos/farmacología , Animales , Apoptosis/genética , Benzodioxoles/farmacología , Caspasa 3/genética , Caspasa 3/metabolismo , Caspasa 9/genética , Caspasa 9/metabolismo , Línea Celular , Citosol/efectos de los fármacos , Citosol/metabolismo , Regulación de la Expresión Génica , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Síndrome Nefrótico/inducido químicamente , Síndrome Nefrótico/metabolismo , Síndrome Nefrótico/patología , Podocitos/metabolismo , Podocitos/patología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Purinas/farmacología , Transducción de Señal , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismoRESUMEN
In order to discover novel derivatives in the anti-tumor field, reported anti-tumor pharmacophores (uridine, uracil, and thymine) were combined with 2-methoxyestradiol, which has been characterized as having excellent biological properties in terms of anti-tumor activity. Thus, 20 hybrids were synthesized through etherification at the 17ß-OH or 3-phenolic hydroxyl group of 2-methoxyestradiol, and evaluated for their biological activities against the human breast adenocarcinoma MCF-7 cell lines, human breast cancer MDA-MB-231 cell lines, and the normal human liver L-O2 cell lines. As a result, all the uridine derivatives and single-access derivatives of uracil/thymine possessed good anti-proliferative activity against tested tumor cells (half maximal inhibitory concentration values from 3.89 to 19.32 µM), while only one dual-access derivative (21b) of thymine possessed good anti-proliferative activity (half maximal inhibitory concentration ≈ 25 µM). Among them, the uridine derivative 11 and the single-access derivative of uracil 12a possessed good anti-proliferative selectivity against tested tumor cells. Furthermore, basic mechanism studies revealed that hybrids 11 and 12a could induce apoptosis in MCF-7 cells through mitochondrial pathway. These hybrids induced morphological changes in MCF-7 cells, causing mitochondrial depolarization. These two hybrids also had the following effects: arrest of the cell cycle at the G2 phase; upregulation of Apaf-1, Bax, and cytochrome c; downregulation of Bcl-2 and Bcl-xL for both mRNA and protein; and increase of the expression for caspase-8 and -9. Finally, apoptotic effector caspase-3 was increased, which eventually caused nuclear apoptosis at least through an intrinsic pathway in the mitochondria. Additionally, hybrids 11 and 12a could specifically bind to estradiol receptor alpha in a dose-dependent manner.
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2-Metoxiestradiol/análogos & derivados , 2-Metoxiestradiol/farmacología , Antineoplásicos/química , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , 2-Metoxiestradiol/síntesis química , Antineoplásicos/síntesis química , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Ciclo Celular/efectos de los fármacos , Línea Celular , Línea Celular Tumoral , Femenino , Humanos , Células MCF-7 , Modelos MolecularesRESUMEN
Discovering the utmost effective and targeted chemotherapy for hepatocellular carcinoma is still a significant challenge. In the present study, diethylnitrosamine was used as a liver carcinogen and boldine a compound of boldo. We anticipated the hypothesis that boldine endow antiproliferative and promote apoptosis on hepatocarcinoma rats. We analyzed that boldine alters the tumor biomarkers and liver markers enzyme levels. Also, we determined boldine modulate the enzymatic and nonenzymatic antioxidant activities, as well as messenger RNA and protein expressions of Bcl2, Bax, and cleaved caspase 3 by reverse transcription polymerase chain reaction and Western blot analysis, respectively. It was also manifested by histopathology studies in liver tissues of HCC rats. Our finding suggested that boldine has antioxidant activity, and moreover, also contributes apoptotic nature by upregulating the protein expression of Bax, and cleaved caspase 3. Our data accomplishes that boldine a candidate drug has dynamic therapeutic activity and suitable for the treatment of HCC.
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Antioxidantes/uso terapéutico , Aporfinas/uso terapéutico , Carcinoma Hepatocelular/inducido químicamente , Carcinoma Hepatocelular/tratamiento farmacológico , Dietilnitrosamina/farmacología , Neoplasias Hepáticas/inducido químicamente , Neoplasias Hepáticas/tratamiento farmacológico , Extractos Vegetales/uso terapéutico , Animales , Apoptosis/efectos de los fármacos , Antígeno Carcinoembrionario/sangre , Caspasa 3/metabolismo , Proliferación Celular/efectos de los fármacos , Citocromos c/metabolismo , Hígado/efectos de los fármacos , Hígado/patología , Masculino , Estrés Oxidativo/efectos de los fármacos , Peumus/química , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , ARN Mensajero/efectos de los fármacos , Ratas , Ratas Wistar , Aumento de Peso , alfa-Fetoproteínas/análisis , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Colorectal cancer (CRC) is a leading killer cancer worldwide and one of the most common malignancies with increasing incidences of mortality. Guggulsterone (GS) is a plant sterol used for treatment of various ailments such as obesity, hyperlipidemia, diabetes, and arthritis. In the current study, anti-cancer effects of GS in human colorectal cancer cell line HCT 116 was tested, potential targets identified using mass spectrometry-based label-free shotgun proteomics approach and key pathways validated by proteome profiler antibody arrays. Comprehensive proteomic profiling identified 14 proteins as significantly dysregulated. Proteins involved in cell proliferation/migration, tumorigenesis, cell growth, metabolism, and DNA replication were downregulated while the protein with functional role in exocytosis/tumor suppression was found to be upregulated. Our study evidenced that GS treatment altered expression of Bcl-2 mediated the mitochondrial release of cytochrome c which triggered the formation of apoptosome as well as activation of caspase-3/7 leading to death of HCT 116 cells via intrinsic apoptosis pathway. GS treatment also induced expression of p53 protein while p21 expression was unaltered with no cell cycle arrest. In addition, GS was found to inhibit NF-kB signaling in colon cancer cells by quelling the expression of its regulated gene products Bcl-2, cIAP-1, and survivin.
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In the past decade, the research communities raised wide concerns on using medicinal plants for synthesis of nanomaterials due to its effective biological activity, lower side effects and also eco-friendly manner. Our previous report concentrated on the biomedical efficacy of fine characterized silver nanoparticles (AgNPs) from Gossypium hirsutum (cotton) leaf extract. Further, the current examination is planned to reveal the molecular mechanisms involving for activation of mitochondria-mediated signaling pathway by AgNPs in human lung cancer cells (A549) using various biological endpoints such as apoptotic induction by HOECHST 33342, AO/EtBr and Rhodamine 123 staining, cell cycle analysis using flow cytometry, gene and protein expressions by RT-PCR and immunoblotting respectively. This study was further extended to identify the toxicity of AgNPs using an animal model. Interestingly, we observed that A549 cells treated with AgNPs resulted in G2/M arrest and ultimately leads to induction of apoptosis cell death. Moreover, gene analysis demonstrated that diminished expression of anti-apoptotic (Bcl-2) and enhanced expression of pro-apoptotic (Bax) mitochondrial genes. The alterations in the gene pattern may interrupt of mitochondrial membrane potential which facilitates the releasing of cytochrome c (cyt c) into cytosol. The cyt c act as a key molecule for activation of caspases (9 and 3) to initiate intrinsic apoptotic signaling cell death process. The histological analysis proven the application of AgNPs in nanomedicine is quietly harmless and would not cause any discernible stress like swelling and inflammation to the organs of mice. Taken together, this investigation may provide solid evidence for cotton crop mediated AgNPs induced apoptosis cell death pathway and offer a novel approach for cancer therapy.
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Amyotrophic lateral sclerosis (ALS) is a common neurodegenerative disorder, but little is known about the exact causes and pathophysiology of this disease. In transgenic mouse models of ALS, mitochondrial abnormalities develop during the disease and might contribute to the progression of ALS. Gene therapy was recently shown to induce beneficial effects. For example, the delivery of human insulin-like growth factor-1 (hIGF-1) by self-complementary adeno-associated virus (AAV) vectors has been shown to prolong the lifespan of ALS transgenic mice. However, the function of IGF-1 in mitochondria has not been systematically studied in ALS models. In this study, scAAV9-hIGF-1 was intramuscularly injected into transgenic SOD1G93A mice and administered to cell lines expressing the â¼25-kDa C-terminal fragment of transactive response DNA-binding protein (TDP-25). The mitochondrial electrical transmembrane potential was hyperpolarized, and electron microscopy findings revealed that the abnormal mitochondria were transformed. Moreover, the intrinsic mitochondrial apoptotic process was modified through the upregulation of anti-apoptotic proteins (B-cell lymphoma-extra large (Bcl-xl) and B-cell lymphoma-2 (Bcl-2)), the downregulation of pro-apoptotic proteins (Bcl-2-associated x protein (Bax) and Bcl-2 homologous antagonist killer (Bak)) and a reduction in mitochondrial cytochrome c release. Mitophagy was also increased after scAAV9-hIGF-1 treatment, as evidenced by a decrease in the p62 level and an increase in the LC3-II level. Furthermore, the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) system was used to delete the IGF-1 gene in SOD1G93A model mice via an intrathecal injection of scAAV9-sgRNA-IGF1-Cas9 to confirm these findings. The protective effect of IGF-1 on the mitochondria decreased after genetic deletion. These novel findings demonstrate that IGF-1 strongly protects mitochondria from apoptosis and upregulates mitophagy in mouse and cell models of ALS. Therefore, therapies that specifically protect mitochondrial function might be promising strategies for treating ALS.
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Esclerosis Amiotrófica Lateral/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Esclerosis Amiotrófica Lateral/fisiopatología , Animales , Apoptosis/fisiología , Línea Celular , Proteínas de Unión al ADN/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Humanos , Factor I del Crecimiento Similar a la Insulina/fisiología , Masculino , Potencial de la Membrana Mitocondrial/fisiología , Ratones , Ratones Transgénicos , Mitocondrias/metabolismo , Mitofagia/fisiología , Neuronas Motoras/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Fragmentos de Péptidos/metabolismo , Superóxido Dismutasa/metabolismoRESUMEN
RATIONALE: ADAM8 (a disintegrin and metalloproteinase domain-8) is expressed by leukocytes and epithelial cells in health, but its contribution to the pathogenesis of chronic obstructive pulmonary disease (COPD) is unknown. OBJECTIVES: To determine whether the expression of ADAM8 is increased in the lungs of patients with COPD and cigarette smoke (CS)-exposed mice, and whether ADAM8 promotes the development of COPD. METHODS: ADAM8 levels were measured in lung, sputum, plasma, and/or BAL fluid samples from patients with COPD, smokers, and nonsmokers, and wild-type (WT) mice exposed to CS versus air. COPD-like lung pathologies were compared in CS-exposed WT versus Adam8-/- mice. MEASUREMENTS AND MAIN RESULTS: ADAM8 immunostaining was reduced in macrophages, and alveolar and bronchial epithelial cells in the lungs of patients with COPD versus control subjects, and CS- versus air-exposed WT mice. ADAM8 levels were similar in plasma, sputum, and BAL fluid samples from patients with COPD and control subjects. CS-exposed Adam8-/- mice had greater airspace enlargement and airway mucus cell metaplasia than WT mice, but similar small airway fibrosis. CS-exposed Adam8-/- mice had higher lung macrophage counts, oxidative stress levels, and alveolar septal cell death rates, but lower alveolar septal cell proliferation rates and soluble epidermal growth factor receptor BAL fluid levels than WT mice. Adam8 deficiency increased lung inflammation by reducing CS-induced activation of the intrinsic apoptosis pathway in macrophages. Human ADAM8 proteolytically shed the epidermal growth factor receptor from bronchial epithelial cells to reduce mucin expression in vitro. Adam8 bone marrow chimera studies revealed that Adam8 deficiency in leukocytes and lung parenchymal cells contributed to the exaggerated COPD-like disease in Adam8-/- mice. CONCLUSIONS: Adam8 deficiency increases CS-induced lung inflammation, emphysema, and airway mucus cell metaplasia. Strategies that increase or prolong ADAM8's expression in the lung may have therapeutic efficacy in COPD.
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Proteínas ADAM/genética , Antígenos CD/genética , Proteínas de la Membrana/genética , Enfermedad Pulmonar Obstructiva Crónica/genética , Enfermedad Pulmonar Obstructiva Crónica/fisiopatología , Anciano , Animales , Fumar Cigarrillos/fisiopatología , Modelos Animales de Enfermedad , Femenino , Humanos , Pulmón/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana EdadRESUMEN
As an indispensable process of cell life, apoptosis is essential for keeping homeostasis at cell level. Dysregulation of apoptosis is usually involved in the pathological processes of many complex diseases including cancer. With the properties such as high affinity and specificity to their targets, easy of synthesis and modification and good biocompatibility, aptamers have been attractive molecules applied in basic research, diagnostics and therapeutics. This review mainly focuses on the recent researches on application of aptamers in interference of cell apoptosis. Key targets along the intrinsic and extrinsic apoptosis pathways were respectively dissected using aptamers as a tool, providing an insight into the pathological processes, especially for cancer.
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Apoptosis/efectos de los fármacos , Aptámeros de Nucleótidos/farmacología , Técnica SELEX de Producción de Aptámeros/métodos , Transducción de Señal/efectos de los fármacos , Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Aptámeros de Nucleótidos/genética , Aptámeros de Nucleótidos/metabolismo , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Humanos , Modelos Biológicos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Transducción de Señal/genéticaRESUMEN
Pro-inflammatory cytokine-induced chondrocyte apoptosis is a primary cause of cartilage destruction in the progression of rheumatoid arthritis (RA). Advanced oxidation protein products (AOPPs), a novel pro-inflammatory mediator, have been confirmed to accumulate in patients with RA. However, the effect of AOPPs accumulation on chondrocyte apoptosis and the associated cellular mechanisms remains unclear. The present study demonstrated that the plasma formation of AOPPs was enhanced in RA rats compared with normal. Then, chondrocyte were treated with AOPPs-modified rat serum albumin (AOPPs-RSA) in vitro. Exposure of chondrocyte to AOPPs activated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and increased expression of NADPH oxidase subunits, which was mediated by receptor for advanced glycation end products (RAGE), but not scavenger receptor CD36. Moreover, AOPPs challenge triggered NADPH oxidase-dependent ROS generation which induced mitochondrial dysfunction and endoplasmic reticulum stress resulted in activation of caspase family that eventually lead to apoptosis. Lastly, blockade of RAGE, instead of CD36, largely attenuated these signals. Our study demonstrated first time that AOPPs induce chondrocyte apoptosis via RAGE-mediated and redox-dependent intrinsic apoptosis pathway in vitro. These data implicates that AOPPs may represent a novel pathogenic factor that contributes to RA progression. Targeting AOPPs-triggered cellular mechanisms might emerge as a promising therapeutic option for patients with RA.
Asunto(s)
Productos Avanzados de Oxidación de Proteínas/genética , Artritis Experimental/genética , Condrocitos/metabolismo , Estrés del Retículo Endoplásmico/genética , Receptor para Productos Finales de Glicación Avanzada/genética , Productos Avanzados de Oxidación de Proteínas/metabolismo , Animales , Apoptosis/genética , Artritis Experimental/metabolismo , Artritis Experimental/patología , Antígenos CD36/genética , Antígenos CD36/metabolismo , Condrocitos/patología , Femenino , Regulación de la Expresión Génica , Humanos , Mitocondrias/metabolismo , Mitocondrias/patología , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Oxidación-Reducción , Cultivo Primario de Células , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Receptor para Productos Finales de Glicación Avanzada/metabolismo , Transducción de SeñalRESUMEN
CONTEXT: The rizoma of Pulsatilla chinensis (Bunge) Regel has been used as a traditional Chinese medicinal herb for thousands of years. Total saponins from P. chinensis can induce the apoptosis of solid cancer cells; however, their activity on chronic myeloid leukemia and the mechanisms remains unknown. OBJECTIVE: To study the activity of total saponins and the main active fractions from P. chinensis saponins on chronic myeloid leukemia, and to illustrate the mechanisms underlying the anticancer activities. MATERIALS AND METHODS: The cytotoxic activity were assayed by MTT; cell cycle arrest and apoptosis were tested by flow cytometry system; changes in the mitochondrial membrane potential were determined using JC-1; and the apoptosis signaling pathway was determined by western blotting. RESULTS: We demonstrated that total P. chinensis saponin displayed cytotoxic activity against K562 cell line. In addition, we identified 23-hydroxybetulinic acid (HBA), pulchinenoside A (PA), and anemoside B4 (AB4) from the total saponins, with the most cytotoxic compound HBA. Glycosylation at C3 and C28 of HBA significantly reduces its cytotoxicity. HBA could promote cell cycle arrest at S phase and induce apoptosis via intrinsic pathway. HBA disrupts mitochondrial membrane potential significantly (p < 0.01) and selectively downregulates the levels of Bcl-2, survivin and upregulates Bax, cytochrome C, cleaved caspase-9 and -3. DISCUSSION AND CONCLUSION: Total saponins from P. chinensis may be effective natural products against human chronic myelogenous leukemia; HBA is one of the bioactive components responsible for its anticancer activity, and could be further investigated as an alternative therapeutic drug for leukemia.