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Article in Chinese | WPRIM | ID: wpr-880796


OBJECTIVE@#To investigate the effect of interleukin-17A (IL-17A) on chemosensitivity of ovarian cancer cells to cisplatin (DDP) and explore the mechanism in light of autophagy regulation.@*METHODS@#Ovarian cancer SKOV3 cells cultured @*RESULTS@#DDP increased the expression of IL-17RA in ovarian cancer SKOV3 cells. Treatment with IL-17A significantly reduced the susceptibility of SKOV3 cells to cisplatin-induced apoptosis (@*CONCLUSIONS@#IL-17A/IL-17RA can decrease chemosensitivity of SKOV3 cells to DDP by upregulating DDP-induced autophagy.

Antineoplastic Agents/therapeutic use , Apoptosis/drug effects , Autophagy/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cisplatin/pharmacology , Drug Resistance, Neoplasm/drug effects , Female , Humans , Interleukin-17/pharmacology , Ovarian Neoplasms/drug therapy , Receptors, Interleukin-17
Einstein (Säo Paulo) ; 18: eAO4560, 2020. graf
Article in English | LILACS | ID: biblio-1101099


ABSTRACT Objective To investigate if ICI 182,780 (fulvestrant), a selective estrogen receptor alpha/beta (ERα/ERβ) antagonist, and G-1, a selective G-protein-coupled receptor (GPER) agonist, can potentially induce autophagy in breast cancer cell lines MCF-7 and SKBr3, and how G-1 affects cell viability. Methods Cell viability in MCF-7 and SKBr3 cells was assessed by the MTT assay. To investigate the autophagy flux, MCF-7 cells were transfected with GFP-LC3, a marker of autophagosomes, and analyzed by real-time fluorescence microscopy. MCF-7 and SKBr3 cells were incubated with acridine orange for staining of acidic vesicular organelles and analyzed by flow cytometry as an indicator of autophagy. Results Regarding cell viability in MCF-7 cells, ICI 182,780 and rapamycin, after 48 hours, led to decreased cell proliferation whereas G-1 did not change viability over the same period. The data showed that neither ICI 182,780 nor G-1 led to increased GFP-LC3 puncta in MCF-7 cells over the 4-hour observation period. The cytometry assay showed that ICI 182,780 led to a higher number of acidic vesicular organelles in MCF-7 cells. G-1, in turn, did not have this effect in any of the cell lines. In contrast, ICI 182,780 and G-1 did not decrease cell viability of SKBr3 cells or induce formation of acidic vesicular organelles, which corresponds to the final step of the autophagy process in this cell line. Conclusion The effect of ICI 182,780 on increasing acidic vesicular organelles in estrogen receptor-positive breast cancer cells appears to be associated with its inhibitory effect on estrogen receptors, and GPER does notseem to be involved. Understanding these mechanisms may guide further investigations of these receptors' involvement in cellular processes of breast cancer resistance.

RESUMO Objetivo Avaliar o efeito dos compostos ICI 182,780 (fulvestranto), um antagonista seletivo dos receptores de estrógeno alfa/beta (REα/REβ), e do G-1, um agonista seletivo de receptores de estrógeno acoplados a proteínas-G (GPER), na possível indução de autofagia em linhagens de câncer de mama MCF-7 e SKBr3, bem como o efeito de G-1 na viabilidade celular. Métodos A viabilidade celular de células MCF-7 e SKBr3 foi avaliada pelo ensaio com MTT. Para investigar a indução da autofagia, células MCF-7 foram transfectadas com GFP-LC3, um marcador de autofagossomos, e analisadas por microscopia de fluorescência em tempo real. As células MCF-7 e SKBr3 foram incubadas com o indicador de compartimentos ácidos laranja de acridina e analisadas por citometria de fluxo como indicativo para autofagia. Resultados Em células MCF-7, o ICI 182,780 e rapamicina após 48 horas levaram à diminuição da viabilidade celular, enquanto o G-1 não alterou a viabilidade no mesmo período de tratamento. Nem o ICI 182,780 e nem o G-1 induziram aumento na pontuação de GFP-LC3 em células MCF-7 até 4 horas. Já os ensaios de citometria de fluxo demonstraram que ICI 182,780 levou ao aumento de compartimentos ácidos em células MCF-7. O G-1 não aumentou estes parâmetros em ambas as linhagens. Por outro lado, ICI 182,780 e G-1 não induziram à redução da viabilidade em células SKBr3 e nem à formação de compartimentos ácidos, como etapa final do processo autofágico. Conclusão O aumento de compartimentos ácidos pelo ICI 182,780 em células de câncer de mama positivas para receptores de estrógeno parece estar associado com seu efeito inibidor de receptores de estrógeno, mas sem o envolvimento de GPER. A compreensão desses mecanismos pode direcionar estudos sobre o envolvimento dos receptores nos processos celulares de resistência do câncer de mama.

Humans , Female , Autophagy/drug effects , Breast Neoplasms/pathology , Breast Neoplasms/drug therapy , Receptors, G-Protein-Coupled/agonists , Estrogen Receptor Antagonists/pharmacology , Fulvestrant/pharmacology , Time Factors , Transfection/methods , Cell Survival/drug effects , Blotting, Western , Reproducibility of Results , Analysis of Variance , Sirolimus/pharmacology , Receptors, G-Protein-Coupled/analysis , Estrogen Receptor alpha/antagonists & inhibitors , Estrogen Receptor beta/antagonists & inhibitors , Cell Proliferation/drug effects , MCF-7 Cells , Flow Cytometry/methods
Acta cir. bras ; 34(11): e201901106, Nov. 2019. tab, graf
Article in English | LILACS | ID: biblio-1054683


Abstract Purpose: To investigate whether GDF11 ameliorates myocardial ischemia reperfusion (MIR) injury in diabetic rats and explore the underlying mechanisms. Methods: Diabetic and non-diabetic rats subjected to MIR (30 min of coronary artery occlusion followed by 120 min of reperfusion) with/without GDF11 pretreatment. Cardiac function, myocardial infarct size, creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH), superoxide dismutase (SOD) 15-F2tisoprostane, autophagosome, LC3II/I ratio and Belcin-1 level were determined to reflect myocardial injury, oxidative stress and autophagy, respectively. In in vitro study, H9c2 cells cultured in high glucose (HG, 30mM) suffered hypoxia reoxygenation (HR) with/without GDF11, hydrogen peroxide (H2O2) and autophagy inhibitor 3-methyladenine (3-MA) treatment, cell injury; oxidative stress and autophagy were assessed. Results: Pretreatment with GDF11 significantly improved cardiac morphology and function in diabetes, concomitant with decreased arrhythmia severity, infarct size, CK-MB, LDH and 15-F2tisoprostane release, increased SOD activity and autophagy level. In addition, GDF11 notably reduced HR injury in H9c2 cells with HG exposure, accompanied by oxidative stress reduction and autophagy up-regulation. However, those effects were completely reversed by H2O2 and 3-MA. Conclusion: GDF11 can provide protection against MIR injury in diabetic rats, and is implicated in antioxidant stress and autophagy up-regulation.

Animals , Male , Autophagy/drug effects , Myocardial Reperfusion Injury/metabolism , Myocardial Reperfusion Injury/drug therapy , Oxidative Stress/drug effects , Diabetes Mellitus, Type 1/metabolism , Growth Differentiation Factors/pharmacology , Reference Values , Superoxide Dismutase/analysis , Cardiotonic Agents/pharmacology , Myocardial Reperfusion Injury/pathology , Up-Regulation/drug effects , Cell Line , Blotting, Western , Reproducibility of Results , Rats, Sprague-Dawley , Streptozocin , Microscopy, Electron, Transmission , Diabetes Mellitus, Experimental/metabolism , Hemodynamics/drug effects , Antioxidants/pharmacology
Braz. j. med. biol. res ; 52(2): e7843, 2019. graf
Article in English | LILACS | ID: biblio-984023


Gastric cancer remains a serious threat to human health worldwide. Kaempferol is a plant-derived flavonoid compound with a wide range of pharmacological activities. This study aimed to investigate the effects of kaempferol on gastric cancer SNU-216 cell proliferation, apoptosis, and autophagy, as well as underlying potential mechanisms. Viability, proliferation, and apoptosis of SNU-216 cells after kaempferol treatment were evaluated using cell counting kit-8 assay, 5-btomo-2′-deoxyuridine incorporation assay, and annexin V-FITC/PI staining, respectively. Quantitative reverse transcription PCR was performed to measure the mRNA expressions of cyclin D1 and microRNA-181a (miR-181a) in SNU-216 cells. Cell transfection was used to down-regulate the expression of miR-181a. The protein expression levels of cyclin D1, bcl-2, bax, caspase 3, caspase 9, autophagy-related gene 7, microtubule-associated protein 1 light chain 3-I (LC3-I), LC3-II, Beclin 1, p62, mitogen-activated protein kinase (MAPK), extracellular regulated protein kinases (ERK), and phosphatidylinositol 3 kinase (PI3K) in SNU-216 cells were detected using western blotting. Results showed that kaempferol significantly suppressed SNU-216 cell viability and proliferation but had no influence on cell apoptosis. Further results suggested that kaempferol significantly induced SNU-216 cell autophagy. The expression of miR-181a in SNU-216 cells after kaempferol treatment was enhanced. Kaempferol significantly inactivated MAPK/ERK and PI3K pathways in SNU-216 cells. Suppression of miR-181a significantly reversed the kaempferol-induced MAPK/ERK and PI3K pathways inactivation in SNU-216 cells. This research demonstrated that kaempferol suppressed proliferation and promoted autophagy of human gastric cancer SNU-216 cells by up-regulating miR-181a and inactivating MAPK/ERK and PI3K pathways.

Humans , Autophagy/drug effects , Stomach Neoplasms/pathology , Apoptosis/drug effects , Kaempferols/pharmacology , Cell Proliferation/drug effects , Cell Line, Tumor
Braz. j. med. biol. res ; 52(11): e8371, 2019. graf
Article in English | LILACS | ID: biblio-1039257


Oxiracetam (ORC) is a commonly used nootropic drug for improving cognition and memory impairments. The therapeutic effect and underlying mechanism of ORC in vascular dementia (VaD) treatment remain unknown. In this study, 3-month-old male Sprague-Dawley rats with permanent bilateral common carotid artery occlusion-induced VaD were treated orally with low (100 mg/kg) or high (200 mg/kg) dose ORC once a day for 4 weeks. The results of the Morris water maze test and Nissl staining showed that ORC treatment significantly alleviated learning and memory deficits and neuronal damage in rats with VaD. Mechanistically, the protein levels of a panel of genes associated with neuronal apoptosis (Bcl-2, Bax) and autophagy (microtubule-associated protein 1 chain 3, Beclin1, p62) were significantly altered by ORC treatment compared with VaD, suggesting a protective role of ORC against VaD-induced neuronal apoptosis and autophagy. Moreover, the Akt/mTOR pathway, which is known to be the upstream signaling governing apoptosis and autophagy, was found to be activated in ORC-treated rats, suggesting an involvement of Akt/mTOR activation in ORC-rendered protection in VaD rats. Taken together, this study demonstrated that ORC may alleviate learning and memory impairments and neuronal damage in VaD rats by altering the expression of apoptosis/autophagy-related genes and activation of the Akt/mTOR signaling pathway in neurons.

Animals , Male , Rats , Pyrrolidines/administration & dosage , Dementia, Vascular/drug therapy , Signal Transduction/physiology , Neuroprotective Agents/administration & dosage , Proto-Oncogene Proteins c-akt/metabolism , Cognitive Dysfunction/drug therapy , Autophagy/drug effects , Dementia, Vascular/physiopathology , Dementia, Vascular/metabolism , Rats, Sprague-Dawley , Apoptosis/drug effects , Maze Learning/drug effects , Disease Models, Animal , TOR Serine-Threonine Kinases/metabolism , Cognitive Dysfunction/physiopathology , Cognitive Dysfunction/metabolism
Biol. Res ; 52: 32, 2019. graf
Article in English | LILACS | ID: biblio-1038783


BACKGROUND: Long non-coding RNA H19 (H19) plays an important role by regulating protein expression in different tissues and organs of the body. However, whether H19 induces hypoxia/reoxygenation (h/R) injury via increase of autophagy in the hepatoma carcinoma cells is unknown. RESULTS: H19 was expressed in the hepatoma carcinoma cells (Hep G2 and HCCLM3 cells) and its expression was most in 8 h/24R. The knockdown of H19 and 3-MA (an autophagy inhibitor) protected against h/R-induced apoptosis, cell damage, the expression of cleaved caspase-3 and cleaved caspase-9, the release of cytochrome c (Cyt c). The knockdown of H19 and 3-MA also decreased the autophagic vesicles (AVs) and the expression of Beclin-1 and the ration of LC3-II/LC3-I, and increased cell viability, the expression of Bcl-2 and P62 and the phosphorylation of PI3K, Akt and mTOR. In addition, chloroquine (CQ, an inhibitor of autophagy flux) markedly decreased formation of autophagy flux (the ration of LC3-II/LC3-I). The results of the knockdown of H19 group were similar to those of the 3-MA (or CQ) group. Rapamycin (a mTOR inhibitor, an autophagy activator) further down-regulated h/R-induced decrease of the phosphorylated PI3K, Akt and mTOR. The knockdown of H19 cancelled the effect of rapamycin. The overexpression of H19 further expanded h/R-induced increase of the ration of LC3-II/LC3-I and decrease of the phosphorylated PI3K, Akt and mTOR. CONCLUSIONS: Our results suggest that the long non-coding RNA H19 induces h/R injury by up-regulation of autophagy via activation of PI3K-Akt-mTOR pathway in the hepatoma carcinoma cells.

Humans , Reperfusion Injury/metabolism , Carcinoma, Hepatocellular/metabolism , RNA, Long Noncoding/metabolism , Liver Neoplasms/metabolism , Hypoxia/metabolism , Oxygen/metabolism , Autophagy/drug effects , Up-Regulation/physiology , Brain Ischemia/metabolism , Apoptosis/physiology , Carcinoma, Hepatocellular/pathology , Liver Neoplasms/pathology
Braz. j. med. biol. res ; 51(5): e6889, 2018. graf
Article in English | LILACS | ID: biblio-889078


2-Methyl-2-butanol (MBT) is a chemical compound from the group of alcohols more specifically pentanols, which has shown an excellent anti-cancer activity in our previous study. However, its mechanism of action remains unclear. The present study was designed to investigate the anti-cancer effect of MBT on human retinoblastoma cells. The results showed that the use of MBT leads to HXO-RB44 cell death but is cytotoxic to normal cells at higher concentrations. It showed a dose- as well as a time-dependent inhibition of HXO-RB44 cells. P27 is a cell cycle inhibitory protein, which plays an important role in cell cycle regulation whereas cyclin-B1 is a regulatory protein involved in mitosis. MBT increased the cell cycle arrest in a dose-dependent manner by augmenting p27 and reducing cyclin B1 expression. Moreover, it also accelerated apoptosis, increased light chain-3 (LC-3) conversion in a dose-dependent manner, and helped to debulk cancerous cells. LC3 is a soluble protein, which helps to engulf cytoplasmic components, including cytosolic proteins and organelles during autophagy from autophagosomes. In order to verify the effect of MBT, bafilomycin A1, an autophagy inhibitor, was used to block the MTB-induced apoptosis and necrosis. Additionally, a specific Akt agonist, SC-79, reversed the MBT-induced cell cycle arrest and autophagy. Thus, from the present study, it was concluded that MBT induced cell cycle arrest, apoptosis and autophagy through the PI3K/Akt pathway in HXO-RB44 cells.

Humans , Antineoplastic Agents/pharmacology , Autophagy/drug effects , Cell Cycle Checkpoints/drug effects , Pentanols/pharmacology , Retinoblastoma/pathology , Blotting, Western , Phosphatidylinositol 3-Kinases/drug effects , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/drug effects , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction , Tumor Cells, Cultured
Braz. j. med. biol. res ; 51(6): e7061, 2018. graf
Article in English | LILACS | ID: biblio-889105


Andrographolide (ANDRO) has been studied for its immunomodulation, anti-inflammatory, and neuroprotection effects. Because brain hypoxia is the most common factor of secondary brain injury after traumatic brain injury, we studied the role and possible mechanism of ANDRO in this process using hypoxia-injured astrocytes. Mouse cortical astrocytes C8-D1A (astrocyte type I clone from C57/BL6 strains) were subjected to 3 and 21% of O2 for various times (0-12 h) to establish an astrocyte hypoxia injury model in vitro. After hypoxia and ANDRO administration, the changes in cell viability and apoptosis were assessed using CCK-8 and flow cytometry. Expression changes in apoptosis-related proteins, autophagy-related proteins, main factors of JNK pathway, ATG5, and S100B were determined by western blot. Hypoxia remarkably damaged C8-D1A cells evidenced by reduction of cell viability and induction of apoptosis. Hypoxia also induced autophagy and overproduction of S100B. ANDRO reduced cell apoptosis and promoted cell autophagy and S100B expression. After ANDRO administration, autophagy-related proteins, S-100B, JNK pathway proteins, and ATG5 were all upregulated, while autophagy-related proteins and s100b were downregulated when the jnk pathway was inhibited or ATG5 was knocked down. ANDRO conferred a survival advantage to hypoxia-injured astrocytes by reducing cell apoptosis and promoting autophagy and s100b expression. Furthermore, the promotion of autophagy and s100b expression by ANDRO was via activation of jnk pathway and regulation of ATG5.

Animals , Mice , Astrocytes/drug effects , Autophagy/drug effects , Cell Hypoxia/drug effects , Diterpenes/pharmacology , S100 Calcium Binding Protein beta Subunit/drug effects , Apoptosis/drug effects , Astrocytes/physiology , Blotting, Western , Cell Survival/drug effects , Real-Time Polymerase Chain Reaction , S100 Calcium Binding Protein beta Subunit/metabolism , Time Factors , Transfection
Clinics ; 73(supl.1): e814s, 2018. tab, graf
Article in English | LILACS | ID: biblio-974944


Cancer is a leading cause of death worldwide, and its incidence is continually increasing. Although anticancer therapy has improved significantly, it still has limited efficacy for tumor eradication and is highly toxic to healthy cells. Thus, novel therapeutic strategies to improve chemotherapy, radiotherapy and targeted therapy are an important goal in cancer research. Macroautophagy (herein referred to as autophagy) is a conserved lysosomal degradation pathway for the intracellular recycling of macromolecules and clearance of damaged organelles and misfolded proteins to ensure cellular homeostasis. Dysfunctional autophagy contributes to many diseases, including cancer. Autophagy can suppress or promote tumors depending on the developmental stage and tumor type, and modulating autophagy for cancer treatment is an interesting therapeutic approach currently under intense investigation. Nutritional restriction is a promising protocol to modulate autophagy and enhance the efficacy of anticancer therapies while protecting normal cells. Here, the description and role of autophagy in tumorigenesis will be summarized. Moreover, the possibility of using fasting as an adjuvant therapy for cancer treatment, as well as the molecular mechanisms underlying this approach, will be presented.

Humans , Autophagy/physiology , Fasting/physiology , Neoplasms/physiopathology , Neoplasms/therapy , Autophagy/drug effects , Autophagy/radiation effects , Antineoplastic Protocols , Neoplasms/metabolism , Antineoplastic Agents/pharmacology
Biol. Res ; 51: 22, 2018. graf
Article in English | LILACS | ID: biblio-950906


BACKGROUND: Our study aimed to investigate the roles of autophagy against high glucose induced response in retinal pigment epithelium (ARPE-19 cells). METHODS: The morphological changes and reactive oxygen species (ROS) generation in ARPE-19 cells under high glucose treatment were respectively detected using the transmission electron microscopy and flow cytometry. The expression levels of Parkin, PINK1, BNIP3L, LC3-I and LC3-II in ARPE-19 cells received high glucose treatment were measured by western blot after pretreatment of carbonyl cyanide m-chlorophenylhydrazone (CCCP), 3-methyladenine (3-MA), N-acetyl cysteine (NAC) or cyclosporin A (CsA) followed by high glucose treatment. RESULTS: ARPE-19 cells subjected to high glucose stress showed an obvious reduction in the LC3-I expression and significant increase in the number of autophagosomes, in the intracellular ROS level, and in the expression levels of Parkin, PINK1, BNIP3L and LC3-II (p < 0.05). Pretreatment with CCCP significantly reduced the LC3-I expression and increased the expression levels of Parkin, PINK1, BNIP3L and LC3-II (p < 0.05). ARPE-19 cells pretreated with CsA under high glucose stress showed markedly down-regulated expressions of Parkin, PINK1 and BNIP3L compared with the cells treated with high glucose (p < 0.05). Pretreatment of ARPE-19 cells with NAC or 3-MA under high glucose stress resulted in a marked reduction in the expression levels of PINK1, BNIP3L and LC3-II (p < 0.05). Meanwhile, the expression level of Parkin in the ARPE-19 cells pretreated with NAC under high glucose stress was comparable with that in the control cells. CONCLUSION: Autophagy might have protective roles against high glucose induced injury in ARPE19 cells via regulating PINK1/Parkin pathway and BNIP3L.

Humans , Protein Kinases/drug effects , Autophagy/drug effects , Proto-Oncogene Proteins/drug effects , Tumor Suppressor Proteins/drug effects , Ubiquitin-Protein Ligases/drug effects , Retinal Pigment Epithelium/drug effects , Glucose/pharmacology , Membrane Proteins/drug effects , Protein Kinases/metabolism , Autophagy/physiology , Signal Transduction/physiology , Cell Line , Proto-Oncogene Proteins/metabolism , Reactive Oxygen Species/metabolism , Tumor Suppressor Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Microscopy, Electron, Transmission , Retinal Pigment Epithelium/cytology , Flow Cytometry , Membrane Proteins/metabolism
An. acad. bras. ciênc ; 89(1): 247-261, Jan,-Mar. 2017. graf
Article in English | LILACS | ID: biblio-886640


ABSTRACT Prosopis juliflora is a shrub that has been used to feed animals and humans. However, a synergistic action of piperidine alkaloids has been suggested to be responsible for neurotoxic damage observed in animals. We investigated the involvement of programmed cell death (PCD) and autophagy on the mechanism of cell death induced by a total extract (TAE) of alkaloids and fraction (F32) from P. juliflora leaves composed majoritary of juliprosopine in a model of neuron/glial cell co-culture. We saw that TAE (30 µg/mL) and F32 (7.5 µg/mL) induced reduction in ATP levels and changes in mitochondrial membrane potential at 12 h exposure. Moreover, TAE and F32 induced caspase-9 activation, nuclear condensation and neuronal death at 16 h exposure. After 4 h, they induced autophagy characterized by decreases of P62 protein level, increase of LC3II expression and increase in number of GFP-LC3 cells. Interestingly, we demonstrated that inhibition of autophagy by bafilomycin and vinblastine increased the cell death induced by TAE and autophagy induced by serum deprivation and rapamycin reduced cell death induced by F32 at 24 h. These results indicate that the mechanism neural cell death induced by these alkaloids involves PCD via caspase-9 activation and autophagy, which seems to be an important protective mechanism.

Animals , Rats , Piperidines/toxicity , Autophagy/physiology , Neuroglia/drug effects , Prosopis/chemistry , Alkaloids/toxicity , Piperidines/isolation & purification , Autophagy/drug effects , Time Factors , Plant Extracts/toxicity , Cell Survival/drug effects , Cells, Cultured , Adenosine Triphosphate/analysis , Neuroglia/physiology , Cell Death/drug effects , Cell Death/physiology , Rats, Wistar , Alkaloids/isolation & purification , Membrane Potential, Mitochondrial/drug effects , Membrane Potential, Mitochondrial/physiology
Egyptian Journal of Hospital Medicine [The]. 2016; 62 (January): 65-76
in English | IMEMR | ID: emr-180261


Backgrounds: Natural remedies were used for cancer treatments, particular breast cancer. Also, the consumption of food products containing high amount of flavonoids and antioxidants had reported to lower the risk of various cancers. Bee venom [BV] and propolis were produced by honey bee. They were characterized by naturopathic formulation, affordability and containing high amount of antioxidants. Moreover, they were used safely since ancient times globally. Although that, there is no information about the synergistic or antagonistic anticancer effects of their combination. This study was designed to evaluate cytotoxic and pro-apoptotic effects of BV, propolis, and their combination on breast cancer [MCF-7] cells

Materials and Methods: As preliminary study, MCF-7 cells were treated with BV [5, 10, and 20micro g/ml] and propolis [50, 150, and 450micro g/ml] to specify the desired combination doses of each treatment with no anticancer effect individually. Consequently, doses of [5micro g/ml BV+ 50micro g/ml propolis and 5micro g/ml BV+ 150micro g/ml propolis] were chosen to evaluate the possible synergistic anticancer potential between them. All groups in this study were examined at 2, 4, and 12 hours intervals. The morphological changes were evaluated by acridine orange/ ethidium bromide dual fluorescent staining and Giemsa staining to reveal the formation of apoptotic bodies or nuclear condensation and cytoplasmic blebbing, respectively. DNA fragmentation assay was also carried out to record the reduction in DNA content and apoptosis. Bcl-2 expression, cytoplasmic anti-apoptotic marker, was used to prove the apoptotic properties, and autophagic cell death by florescent microscopy was evaluated also

Results: Morphological observation by inverted and florescent microscopy revealed apoptotic cell death under exposure to BV [10 and 20micro g/ml] and propolis [450micro g/ml]. On the other hand, the results of combined treatments revealed significant morphological alterations after fluorescent and Giemsa staining. Apoptotic DNA fragmentation was clearly observed and Bcl-2 recoded significant down regulation which proved the apoptotic properties of combined treatments. Additionally, autophagic degradation results also supported the occurrence of stress on treated cells leading finally to cell death. All results of powerful anticancer potential were obvious among all combined-treated groups in dose and time dependent manner. This clear that, the combined treatments have possible synergistic effect which, propose it as potential candidates to be used in development of chemotherapy

Humans , MCF-7 Cells , Adenocarcinoma/drug therapy , Antineoplastic Combined Chemotherapy Protocols , Bee Venoms/pharmacology , Propolis/pharmacology , Autophagy/drug effects , Apoptosis/drug effects
Rev. bras. epidemiol ; 18(3): 552-567, Jul.-Sep. 2015. tab
Article in English | LILACS | ID: lil-756007



Viral hepatitis is an important public health problem in Brazil and around the world.


To evaluate vaccination coverage against hepatitis B in adolescents and to identify the associated factors and reasons for non-adherence.


A cross-sectional population-based study with sampling by clusters and in two stages, carried out from records of 702 adolescents aged 11 to 19 years old, non-institutionalized, living in an urban area of Campinas, São Paulo, Brazil, in 2008/2009. The data were obtained from the Health Survey in the city of Campinas (ISACamp).


The prevalence of vaccination (3 doses) was 72.2%. An independent and negative association with the vaccine was observed for the adolescents who were not born in the municipality. The orientation of a health care provider was positively and significantly associated with vaccination. The main reasons for non-adherence were the lack of orientation and not considering the vaccine necessary. Socioeconomic factors, health behaviors and conditions did not restrict the access to vaccination, but the coverage was below the target established by the Ministry of Health in Brazil.


Health education programs, addressing the importance of vaccination to prevent the disease; strategies to actively reach out adolescents that did not complete the schedule; as well as orientation from the health care professional about the benefits of the vaccine to the adolescents, parents and guardians can extend the vaccination coverage.



As hepatites virais constituem importante problema de saúde pública no Brasil e em todo o mundo.


Avaliar a cobertura vacinal contra hepatite B em adolescentes e identificar os fatores associados e motivos da não adesão.


Estudo transversal de base populacional com amostra por conglomerados e em 2 estágios realizado a partir de 702 registros de adolescentes com idade entre 11 e 19 anos, não institucionalizados, residentes em área urbana no município de Campinas, São Paulo, em 2008/2009. Os dados foram obtidos do Inquérito de Saúde no município de Campinas (ISACamp).


A prevalência de vacinação (3 doses) foi de 72,2%. Associação independente e negativa com a vacina foi observada para os adolescentes não naturais do município. A orientação de profissional de saúde esteve positiva e fortemente associada à vacinação. Os principais motivos para a não adesão foram a falta de orientação e não considerar a vacina necessária. Condições socioeconômicas, comportamentos e condições de saúde não restringiram o acesso à vacinação, mas a cobertura esteve abaixo da meta estabelecida pelo Ministério da Saúde.


Programas de educação em saúde, abordando a importância da vacinação na prevenção da doença, estratégias para busca ativa aos adolescentes que não completaram o esquema, bem como a orientação do profissional de saúde sobre os benefícios da vacina aos adolescentes, pais e responsáveis podem ampliar as coberturas vacinais.


Humans , Autophagy/drug effects , Chloroquine/pharmacology , Oncogenes , ras Proteins/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Lung Neoplasms/pathology , Phagosomes/drug effects , Phagosomes/metabolism , /metabolism
Rev. méd. Chile ; 143(2): 237-243, feb. 2015. ilus
Article in Spanish | LILACS | ID: lil-742575


Currently, there is no discussion on the need to improve and strengthen the institutional health care modality of FONASA (MAI), the health care system used by the public services net and by most of the population, despite the widely known and long lasting problems such as waiting lists, hospital debt with suppliers, lack of specialists and increasing services purchase transference to the private sector, etc. In a dichotomous sectorial context, such as the one of health’s social security in Chile (the state on one side and the market on the other), points of view are polarized and stances tend to seek refuge within themselves. As a consequence, to protect the public solution is commonly associated with protecting the “status quo”, creating an environment that is reluctant to change. The author proposes a solution based on three basic core ideas, which, if proven effective, can strengthen each other if combined properly. These are: network financing management, governance of health care services in MAI and investments and human resources in networked self-managed institutions. The proposal of these core ideas was done introducing a reality testing that minimizes the politic complexity of their implementation.

Animals , Humans , Rats , AMP-Activated Protein Kinases/metabolism , Antioxidants/therapeutic use , Autophagy/drug effects , Signal Transduction/drug effects , Sirtuin 1/metabolism , Stilbenes/therapeutic use , Cell Line, Transformed , Dose-Response Relationship, Drug , Doxycycline/pharmacology , Gene Expression Regulation/drug effects , Insecticides/toxicity , Microscopy, Immunoelectron/methods , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Mutation/genetics , Poly(ADP-ribose) Polymerases/metabolism , RNA, Small Interfering/pharmacology , Rotenone/toxicity , Time Factors , alpha-Synuclein/genetics , alpha-Synuclein/metabolism
Article in English | WPRIM | ID: wpr-57308


Mammalian cells remove misfolded proteins using various proteolytic systems, including the ubiquitin (Ub)-proteasome system (UPS), chaperone mediated autophagy (CMA) and macroautophagy. The majority of misfolded proteins are degraded by the UPS, in which Ub-conjugated substrates are deubiquitinated, unfolded and cleaved into small peptides when passing through the narrow chamber of the proteasome. The substrates that expose a specific degradation signal, the KFERQ sequence motif, can be delivered to and degraded in lysosomes via the CMA. Aggregation-prone substrates resistant to both the UPS and the CMA can be degraded by macroautophagy, in which cargoes are segregated into autophagosomes before degradation by lysosomal hydrolases. Although most misfolded and aggregated proteins in the human proteome can be degraded by cellular protein quality control, some native and mutant proteins prone to aggregation into beta-sheet-enriched oligomers are resistant to all known proteolytic pathways and can thus grow into inclusion bodies or extracellular plaques. The accumulation of protease-resistant misfolded and aggregated proteins is a common mechanism underlying protein misfolding disorders, including neurodegenerative diseases such as Huntington's disease (HD), Alzheimer's disease (AD), Parkinson's disease (PD), prion diseases and Amyotrophic Lateral Sclerosis (ALS). In this review, we provide an overview of the proteolytic pathways in neurons, with an emphasis on the UPS, CMA and macroautophagy, and discuss the role of protein quality control in the degradation of pathogenic proteins in neurodegenerative diseases. Additionally, we examine existing putative therapeutic strategies to efficiently remove cytotoxic proteins from degenerating neurons.

Alzheimer Disease/drug therapy , Amyloid beta-Peptides/metabolism , Amyotrophic Lateral Sclerosis/drug therapy , Animals , Autophagy/drug effects , DNA-Binding Proteins/metabolism , Humans , Huntington Disease/drug therapy , Lysosomes/metabolism , Molecular Targeted Therapy , Mutation , Nerve Tissue Proteins/genetics , Neurodegenerative Diseases/drug therapy , Parkinson Disease/drug therapy , PrPSc Proteins/metabolism , Prion Diseases/drug therapy , Proteasome Endopeptidase Complex/metabolism , Proteolysis , Proteostasis Deficiencies/metabolism , Superoxide Dismutase/metabolism , Ubiquitin/metabolism , alpha-Synuclein/metabolism , tau Proteins/metabolism
Indian J Exp Biol ; 2014 Apr; 52(4): 295-304
Article in English | IMSEAR | ID: sea-150359


Natural autophagy and autophagic cell death is being studied in the model system, D. discoideum, which has well known genetic and experimental advantages over the other known systems. There is no apoptotic machinery present in this organism which could interfere with the non-apoptotic cell death. The target of rapamycin (TOR) pathway is a major nutrient-sensing pathway which when inhibited by the drug rapamycin induces autophagy. Rapamycin was originally discovered as an anti-fungal agent but its use was abandoned when it was discovered to have potent immunosuppressive and anti-proliferative properties. It is a known drug used today for various cancer treatments and also for increasing longevity in many model organisms. It has a wide usage but its effects on other pathways or molecules are not known. This model system was used to study the action of rapamycin on autophagy induction. Using the GFP-Atg8, an autophagosome marker, it was shown that rapamycin treatment can induce autophagy by an accumulation of reactive oxygen species and intracellular free calcium. Rapamycin suppresses proliferation by induction of cell cycle arrest in the G1 phase. Taken together, the results suggest that the core machinery for autophagy is conserved in D. discoideum and it can serve as a good model system to delineate the action of rapamycin induced autophagy.

Antioxidants/metabolism , Autophagy/drug effects , Calcium/metabolism , Cell Survival/drug effects , Cells, Cultured , Dictyostelium/drug effects , Dictyostelium/physiology , G1 Phase/drug effects , Oxidative Stress/drug effects , Reactive Oxygen Species/metabolism , Sirolimus/pharmacology
Article in English | WPRIM | ID: wpr-113785


Autophagy is a conserved lysosomal self-digestion process used for the breakdown of long-lived proteins and damaged organelles, and it is associated with a number of pathological processes, including cancer. Phospholipase D (PLD) isozymes are dysregulated in various cancers. Recently, we reported that PLD1 is a new regulator of autophagy and is a potential target for cancer therapy. Here, we investigated whether PLD2 is involved in the regulation of autophagy. A PLD2-specific inhibitor and siRNA directed against PLD2 were used to treat HT29 and HCT116 colorectal cancer cells, and both inhibition and genetic knockdown of PLD2 in these cells significantly induced autophagy, as demonstrated by the visualization of light chain 3 (LC3) puncta and autophagic vacuoles as well as by determining the LC3-II protein level. Furthermore, PLD2 inhibition promoted autophagic flux via the canonical Atg5-, Atg7- and AMPK-Ulk1-mediated pathways. Taken together, these results suggest that PLD2 might have a role in autophagy and that its inhibition might provide a new therapeutic basis for targeting autophagy.

Autophagy/drug effects , Cell Line, Tumor , Colorectal Neoplasms/enzymology , Genetic Therapy , HCT116 Cells , Humans , Phospholipase D/antagonists & inhibitors , Quinolines/pharmacology , RNA Interference , RNA, Small Interfering/genetics , Signal Transduction/drug effects , Spiro Compounds/pharmacology
Biol. Res ; 46(2): 189-200, 2013. ilus, graf, tab
Article in English | LILACS | ID: lil-683997


Lycopene is common in diet and known for its antioxidant activities. However, the impact of lycopene on iron metabolism is poorly investigated. In this study, we hypothesize that lycopene can prevent iron-mediated oxidative stress, proliferation and autophagy in liver and use a rat model of nutritional iron supplementation to confirm its intervention in these defence mechanisms. We found that iron supplementation induced cell proliferation predominantly in non parenchymal cells compared with hepatocytes, but not apoptosis. In addition, iron was accumulated within the hepatic lysosomes where it triggered autophagy as evidenced by the formation of autophagic vesicles detected by LC3-B staining. Iron supplementation also induced morphologic alterations of the mitochondrial membranes probably due to increased lipid peroxidation as indicated by elevated iron and malondialdehyde concentrations in serum and tissues. Lycopene reduced iron-catalyzed lipid peroxidation by decreasing the malondialdehyde level in the liver and colon and enhancing the total superoxide dismutase activities in serum and tissues. The result suggest that lycopene prevents iron-induced oxidative stress, proliferation and autophagy at both biochemical and histological levels due to its potent free radical scavenging and antioxidant properties.

Animals , Male , Antioxidants/administration & dosage , Autophagy/drug effects , Carotenoids/administration & dosage , Cell Proliferation/drug effects , Iron/adverse effects , Oxidative Stress/drug effects , Apoptosis/drug effects , Hepatocytes/drug effects , Hepatocytes/ultrastructure , Iron/blood , Liver/drug effects , Rats, Wistar
Article in English | WPRIM | ID: wpr-158223


The accumulation of abnormal protein aggregates is a major characteristic of many neurodegenerative disorders, including Parkinson's disease (PD). The intracytoplasmic deposition of alpha-synuclein aggregates and Lewy bodies, often found in PD and other alpha-synucleinopathies, is thought to be linked to inefficient cellular clearance mechanisms, such as the proteasome and autophagy/lysosome pathways. The accumulation of alpha-synuclein aggregates in neuronal cytoplasm causes numerous autonomous changes in neurons. However, it can also affect the neighboring cells through transcellular transmission of the aggregates. Indeed, a progressive spreading of Lewy pathology among brain regions has been hypothesized from autopsy studies. We tested whether inhibition of the autophagy/lysosome pathway in alpha-synuclein-expressing cells would increase the secretion of alpha-synuclein, subsequently affecting the alpha-synuclein deposition in and viability of neighboring cells. Our results demonstrated that autophagic inhibition, via both pharmacological and genetic methods, led to increased exocytosis of alpha-synuclein. In a mixed culture of alpha-synuclein-expressing donor cells with recipient cells, autophagic inhibition resulted in elevated transcellular alpha-synuclein transmission. This increase in protein transmission coincided with elevated apoptotic cell death in the recipient cells. These results suggest that the inefficient clearance of alpha-synuclein aggregates, which can be caused by reduced autophagic activity, leads to elevated alpha-synuclein exocytosis, thereby promoting alpha-synuclein deposition and cell death in neighboring neurons. This finding provides a potential link between autophagic dysfunction and the progressive spread of Lewy pathology.

Adenine/analogs & derivatives , Animals , Autophagy/drug effects , Cell Line , Exocytosis/drug effects , Extracellular Space/metabolism , Humans , Mice , Mice, Knockout , Microtubule-Associated Proteins/deficiency , Phagosomes/drug effects , Protein Structure, Quaternary , Protein Transport/drug effects , alpha-Synuclein/chemistry
Gut and Liver ; : 739-746, 2013.
Article in English | WPRIM | ID: wpr-209550


BACKGROUND/AIMS: Heat shock protein (HSP) 70 is constitutively overexpressed in pancreatic cancer cells (PCCs) and appears to confer protection against chemotherapeutics. We investigated whether modulating HSP 70 increases chemoresponsiveness to gemcitabine in PCCs. METHODS: Varying concentrations of quercetin and gemcitabine, either alone or in combination, were added to PCCs (Panc-1 and MiaPaCa-2). MTT assay was performed to analyze cell viability. HSP 70 expression was assessed by Western blot analysis. Apoptosis was determined by measuring caspase-3 activity. Western blot for the LC3-II protein detected the presence of autophagy. RESULTS: HSP 70 levels were not affected by the incubation of Panc-1 and MiaPaCa-2 cells with gemcitabine, whereas with quercetin, the levels were reduced in both cell lines. The viability of both Panc-1 and MiaPaCa-2 cells significantly decreased with gemcitabine treatment but not with quercetin. A combination of gemcitabine and quercetin decreased the viability of both cell lines in a dose-dependent manner, which was more pronounced than gemcitabine treatment alone. Treatment with either gemcitabine or quercetin augmented caspase-3 activity in both cell lines, and a combination of these compounds further potentiated caspase-3 activity. LC3-II protein expression was negligible with gemcitabine treatment but marked with quercetin. The addition of gemcitabine to quercetin did not potentiate LC3-II protein expression. CONCLUSIONS: Modulation of HSP 70 expression with quercetin enhanced the chemoresponsiveness of PCCs to gemcitabine. The mechanism of cell death was both apoptosis and autophagy.

Antineoplastic Combined Chemotherapy Protocols/pharmacology , Apoptosis/drug effects , Autophagy/drug effects , Caspase 3/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Deoxycytidine/analogs & derivatives , Drug Resistance, Neoplasm/drug effects , HSP70 Heat-Shock Proteins/metabolism , Humans , Microtubule-Associated Proteins/metabolism , Pancreatic Neoplasms/drug therapy , Quercetin/pharmacology