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2.
J Cancer Res Clin Oncol ; 145(10): 2507-2517, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31485766

RESUMO

BACKGROUND: Autophagy plays an important role in regulating cisplatin (CDDP) resistance in gastric cancer cells. However, the underlying mechanism of methioninase (METase) in the regulation of autophagy and CDDP resistance of gastric cancer cells is still not clear. MATERIALS AND METHODS: Western blot was used to detect the levels of autophagy-related proteins, multidrug-resistant 1 (MDR-1), and FoxM1 protein. LncRNA HULC was detected by qRT-PCR. Cell viability was detected using CCK-8 assay. The interaction between lncRNA HULC and FoxM1 was confirmed by RNA pull-down and RIP assay. RESULTS: Lentiviral vector carrying METase (LV-METase) suppressed autophagy and CDDP resistance of drug-resistant gastric cancer cells. LncRNA HULC was significantly downregulated in drug-resistant gastric cancer cells transfected with LV-METase. Besides, we found that lncRNA HULC interacted with FoxM1. In addition, METase suppressed autophagy to reduce CDDP resistance of drug-resistant gastric cancer cells through regulating HULC/FoxM1, and interfering HULC suppressed autophagy to reduce CDDP resistance of drug-resistant gastric cancer cells through regulating FoxM1. Finally, interfering HULC inhibited tumor growth in vivo. CONCLUSION: METase suppressed autophagy to reduce CDDP resistance of drug-resistant gastric cancer cells through regulating HULC/FoxM1 pathway.


Assuntos
Autofagia/efeitos dos fármacos , Autofagia/genética , Resistencia a Medicamentos Antineoplásicos/genética , Proteína Forkhead Box M1/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , RNA Longo não Codificante/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Cisplatino/farmacologia , Modelos Animais de Doenças , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Ligação Proteica , Neoplasias Gástricas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Yonsei Med J ; 60(9): 842-853, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31433582

RESUMO

PURPOSE: Long intergenic non-protein coding RNA 665 (LINC00665) plays a vital role in the development of cancer. Its function in hepatocellular carcinoma (HCC), however, remains largely unknown. MATERIALS AND METHODS: The expressions of LINC00665, miR-186-5p, and MAP4K3 were determined by qRT-PCR. Cell viability and apoptosis were evaluated by MTT and flow cytometry, respectively. Autophagic puncta formation was observed by fluorescence microscopy. Bioinformatics analysis, luciferase reporter assay, RNA immunoprecipitation, and RNA pulldown were performed to identify associations among LINC00665, miR-186-5p, and MAP4K3. Western blot was utilized to examine the expressions of MAP4K3, Beclin-1, and LC3. Tumor growth was evaluated in a xenograft model. RESULTS: Elevations in LINC00665 were observed in HCC tissues and cells. The overall survival of HCC patients with high levels of LINC00665 was shorter than those with low levels. In vitro, LINC00665 depletion inhibited viability and induced apoptosis and autophagy. miR-186-5p interacted with LINC00665 and was downregulated in HCC tissues and cells. Upregulation of miR-186-5p inhibited viability and induced apoptosis and autophagy, which were attenuated by upregulation of LINC00665. MAP4K3 was found to possess binding sites with miR-186-5p and was upregulated in HCC tissues and cells. MAP4K3 depletion inhibited viability and induced apoptosis and autophagy, which were attenuated by miR-186-5p inhibitor. In vivo, miR-186-5p expression was negatively correlated with LINC00665 or MAP4K3 in HCC tissues, while LINC00665 was positively correlated with MAP4K3. LINC00665 knockdown suppressed tumor growth. CONCLUSION: LINC00665 was involved in cell viability, apoptosis, and autophagy in HCC via miR-186-5p/MAP4K3 axis, which may provide a new approach for HCC treatment.


Assuntos
Apoptose/genética , Autofagia/genética , Carcinoma Hepatocelular/patologia , Sobrevivência Celular/genética , Regulação Neoplásica da Expressão Gênica/genética , Neoplasias Hepáticas/patologia , Adulto , Idoso , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação para Baixo , Feminino , Humanos , Neoplasias Hepáticas/genética , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , Proteínas Serina-Treonina Quinases , RNA Longo não Codificante/genética , Regulação para Cima
4.
BMC Plant Biol ; 19(1): 326, 2019 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-31324141

RESUMO

BACKGROUND: Autophagy is a conserved, highly-regulated catabolic process that plays important roles in growth, development and innate immunity in plants. In this study, we compared the rate of autophagy induction in Nicotiana benthamiana plants infected with Tobacco mosaic virus or the TMV 24A + UPD mutant variant, which replicates at a faster rate and induces more severe symptoms. Using a BirA* tag and proximity-dependent biotin identification (BioID) analysis, we identified host proteins that interact with the core autophagy protein, ATG8 in TMV 24A + UPD infected plants. By combining the use of a fast replicating TMV mutant and an in vivo protein-protein screening technique, we were able to gain functional insight into the role of autophagy in a compatible virus-host interaction. RESULTS: Our study revealed an increased autophagic flux induced by TMV 24A + UPD, as compared to TMV in N. benthamiana. Analysis of the functional proteome associated with ATG8 revealed a total of 67 proteins, 16 of which are known to interact with ATG8 or its orthologs in mammalian and yeast systems. The interacting proteins were categorized into four functional groups: immune system process, response to ROS, sulphur amino acid metabolism and calcium signalling. Due to the presence of an ubiquitin-associated (UBA) domain, which is demonstrated to interact with ATG8, the Huntingtin-interacting protein K-like (HYPK) was selected for validation of the physical interaction and function. We used yeast two hybrid (Y2H), bimolecular fluorescence complementation (BiFC) and subcellular localization to validate the ATG8-HYPK interaction. Subsequent down-regulation of ATG8 by virus-induced gene silencing (VIGS) showed enhanced TMV symptoms, suggesting a protective role for autophagy during TMV 24A + UPD infection. CONCLUSION: This study presents the use of BioID as a suitable method for screening ATG8 interacting proteins in planta. We have identified many putative binding partners of ATG8 during TMV 24A + UPD infection in N. benthamiana plants. In addition, we have verified that NbHYPK is an interacting partner of ATG8. We infer that autophagy plays a protective role in TMV 24A + UPD infected plants.


Assuntos
Família da Proteína 8 Relacionada à Autofagia/metabolismo , Doenças das Plantas/virologia , Proteínas de Plantas/metabolismo , Tabaco/virologia , Autofagossomos/metabolismo , Autofagia/genética , Autofagia/fisiologia , Biotinilação , Imunidade Vegetal , Tabaco/metabolismo , Vírus do Mosaico do Tabaco
5.
DNA Cell Biol ; 38(8): 857-864, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31237446

RESUMO

Hepatocellular carcinoma (HCC) has been reported to be one of the major tumors in the world. There is a study indicating that MCM3AP-AS1 is an oncogenic factor in HCC; however, the mechanism by which MCM3AP-AS1 regulates HCC remains not fully understood. Reverse Transcription-quantitative PCR and Western blot approaches were used to detect mRNA and protein levels of various genes. To examine invasion of HCC cells and lymphatic vessel formation of human dermal lymphatic endothelial cells (HDLECs), we employed transwell invasion assay and lymphatic vessel assay. Bioinformatic analysis and luciferase reporter assay were used to establish direct interactions between MCM3AP-AS1 and miR-455. Besides, The Cancer Genome Atlas analyses of HCCs were performed to determine the association of MCM3AP-AS1 and epidermal growth factor receptor (EGFR) with overall survival. MCM3AP-AS1 knockdown impaired invasion of HCC cells and lymphatic vessel formation of HDLECs. MCM3AP-AS1 directly interacted with miR-455. Furthermore, miR-455 inhibitor-transfected HepG2 cells enhanced the invasion and lymphatic vessel formation abilities. The rescue experiments indicated that EGFR was critical for MCM3AP-AS1- and miR-455-regulated invasion and lymphatic vessel formation. More interestingly, autophagy-related genes (Beclin1, LC3 II/I, and ATG7) were abnormally regulated in miR-455 mimic or inhibitor HepG2 cells. miR-455 mimic inhibited cell invasion and lymphatic vessel formation, which was evidently abrogated by ATG7 overexpression. Finally, we analyzed The Cancer Genome Atlas data sets to test the upregulated expression levels of MCM3AP-AS1 and EGFR. In addition, the results showed that low levels of both genes facilitate survival of HCC patients. In this study, we reveal a novel mechanism underlying MCM3AP-AS1-induced HCC metastasis by regulating miR-455. The conclusions provide more insights into understanding mechanism underlying HCC and help development of therapeutical approaches for treating HCC.


Assuntos
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , MicroRNAs/genética , RNA Longo não Codificante/genética , Autofagia/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Receptores ErbB/genética , Receptores ErbB/metabolismo , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Hepáticas/patologia , Vasos Linfáticos/patologia , MicroRNAs/metabolismo , RNA Longo não Codificante/metabolismo
6.
Genes Dev ; 33(11-12): 610-619, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-31160394

RESUMO

Macroautophagy (referred to here as autophagy) degrades and recycles cytoplasmic constituents to sustain cellular and mammalian metabolism and survival during starvation. Deregulation of autophagy is involved in numerous diseases, such as cancer. Cancers up-regulate autophagy and depend on it for survival, growth, and malignancy in a tumor cell-autonomous fashion. Recently, it has become apparent that autophagy in host tissues as well as the tumor cells themselves contribute to tumor growth. Understanding how autophagy regulates metabolism and tumor growth has revealed new essential tumor nutrients, where they come from, and how they are supplied and used, which can now be targeted for cancer therapy.


Assuntos
Autofagia , Neoplasias/metabolismo , Animais , Autofagia/genética , Humanos , Neoplasias/fisiopatologia , Neoplasias/terapia
7.
Mol Med Rep ; 19(6): 4743-4752, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31059030

RESUMO

Interleukin 17A (IL­17A) exerts pleiotropic effects on periodontitis, partially through enhancement of alveolar bone loss. Osteoclasts are the main culprits that absorb alveolar bone. However, studies describing the correlation between IL­17A and osteoclasts are not conclusive. Previously, autophagy was revealed to be involved in osteoclast differentiation and bone resorption. However, the role of autophagy in IL­17A­mediated osteoclast formation is yet to be clarified. In the present study, bone marrow macrophages (BMMs) were treated with or without IL­17A. 3­Methyladenine (3­MA) was applied to inhibit autophagy. Osteoclast formation was detected by tartrate­resistant acid phosphatase (TRAP) staining, immunofluorescence, and scanning electron microscope. The effects of IL­17A on osteoclast­specific genes and autophagy­related genes during osteoclast differentiation were examined by real­time quantitative polymerase chain reaction and western blot analysis. Autophagosomes were observed by transmission electron microscope. Hematoxylin and eosin (H&E), and TRAP staining was adopted to assess alveolar bone destruction and the number of osteoclasts, respectively in a rat periodontitis model. Consequently, IL­17A stimulated osteoclast differentiation and bone resorption of BMMs accompanied by an increase in the mRNA expression of osteoclast­specific genes. Furthermore, IL­17A increased the levels of autophagy­related genes and proteins, and inhibition of autophagy with 3­MA attenuated the IL­17A­mediated osteoclastogenesis. In addition, there was an increase in the number of osteoclasts and alveolar bone resorption with IL­17A treatment in the periodontitis rat model. Collectively, these findings indicated that IL­17A facilitated osteoclast differentiation and bone resorption in vitro and in vivo, which may contribute to the understanding of the molecular basis of IL­17A in alveolar bone destruction and provide insight on the clinical therapeutic targets for periodontitis.


Assuntos
Autofagia/efeitos dos fármacos , Medula Óssea/metabolismo , Reabsorção Óssea/metabolismo , Diferenciação Celular/efeitos dos fármacos , Interleucina-17/farmacologia , Macrófagos/metabolismo , Osteoclastos/metabolismo , Actinas/metabolismo , Adenina/análogos & derivados , Adenina/antagonistas & inibidores , Perda do Osso Alveolar/tratamento farmacológico , Perda do Osso Alveolar/patologia , Animais , Autofagossomos/patologia , Autofagia/genética , Medula Óssea/efeitos dos fármacos , Medula Óssea/patologia , Reabsorção Óssea/patologia , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Osteoclastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Periodontite/tratamento farmacológico , Periodontite/patologia , Ratos , Ratos Sprague-Dawley
8.
Int J Mol Sci ; 20(9)2019 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-31083536

RESUMO

Autophagy is an evolutionarily conserved process in eukaryotes to maintain cellular homeostasis under environmental stress. Intracellular control is exerted to produce energy or maintain intracellular protein quality controls. Autophagy plays an important role in embryogenesis, implantation, and maintenance of pregnancy. This role includes supporting extravillous trophoblasts (EVTs) that invade the decidua (endometrium) until the first third of uterine myometrium and migrate along the lumina of spiral arterioles under hypoxic and low-nutrient conditions in early pregnancy. In addition, autophagy inhibition has been linked to poor placentation-a feature of preeclamptic placentas-in a placenta-specific autophagy knockout mouse model. Studies of autophagy in human placentas have revealed controversial results, especially with regard to preeclampsia and gestational diabetes mellitus (GDM). Without precise estimation of autophagy flux, wrong interpretation would lead to fixed tissues. This paper presents a review of the role of autophagy in pregnancy and elaborates on the interpretation of autophagy in human placental tissues.


Assuntos
Autofagia , Animais , Autofagia/genética , Feminino , Humanos , Modelos Biológicos , Placentação , Gravidez , Complicações na Gravidez/patologia , Reprodução
9.
Nat Cell Biol ; 21(5): 614-626, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31036939

RESUMO

Cell growth is controlled by a lysosomal signalling complex containing Rag small GTPases and mammalian target of rapamycin complex 1 (mTORC1) kinase. Here, we carried out a microscopy-based genome-wide human short interfering RNA screen and discovered a lysosome-localized G protein-coupled receptor (GPCR)-like protein, GPR137B, that interacts with Rag GTPases, increases Rag localization and activity, and thereby regulates mTORC1 translocation and activity. High GPR137B expression can recruit and activate mTORC1 in the absence of amino acids. Furthermore, GPR137B also regulates the dissociation of activated Rag from lysosomes, suggesting that GPR137B controls a cycle of Rag activation and dissociation from lysosomes. GPR137B-knockout cells exhibited defective autophagy and an expanded lysosome compartment, similar to Rag-knockout cells. Like zebrafish RagA mutants, GPR137B-mutant zebrafish had upregulated TFEB target gene expression and an expanded lysosome compartment in microglia. Thus, GPR137B is a GPCR-like lysosomal regulatory protein that controls dynamic Rag and mTORC1 localization and activity as well as lysosome morphology.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Genoma Humano/genética , Proteínas Monoméricas de Ligação ao GTP/genética , Receptores Acoplados a Proteínas-G/genética , Animais , Autofagia/genética , Regulação da Expressão Gênica/genética , Humanos , Lisossomos/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Microglia/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , RNA Interferente Pequeno/genética , Receptores Acoplados a Proteínas-G/antagonistas & inibidores , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento
10.
Fish Shellfish Immunol ; 91: 194-201, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31108175

RESUMO

In mammals, STAT3 (Signal transducer and activator of transcription 3) plays an absolutely vital role in response to cytokines and growth factors. In mammals, IL-6/JAK/STAT3 pathway is closely linked to immune response and promotes cell proliferation, survival and metastasis. Some recent studies have already demonstrated that STAT3 regulates autophagy. As a downstream target gene of STAT3, Bcl-2 (B-cell lymphoma 2) not only participates in regulating apoptosis, but also responds to autophagy. STAT3 regulates autophagy through Bcl-2. In general, the generation of autophagy is always accompanied by the change of apoptosis, and the occurrence of apoptosis is often accompanied by the decreased of cell viability. In grass carp (Ctenopharyngodon idella), LPS-induced autophagy is involved in the release of pro-inflammatory cytokines. However, only the relationship between autophagy and cytokines was illustrated, in which the signaling pathways were not discussed. In the present study, we found that the autophagy inducer, Tunicamycin (Tm), can induce C.Idella Kidney cells (CIK) autophagy. When the cells were incubated with the recombinant human IL-6 (rIL-6) for a short period of times, the mRNA expression level of C.Idella IL-6R and STAT3 were increased. At the same time, the number of GFP-LC3 puncta and the ratio of LC3-II/LC3-I were both decreased obviously in cells. It indicated that the rIL-6 can significantly alleviate autophagy induced by Tm. We speculated that CiSTAT3 may play a key role in the process. To confirm this hypothesis, we performed a rIL-6 activating CiSTAT3 assay. The result demonstrated that rIL-6 can induce CiSTAT3 to form homologous dimmer. The activated CiSTAT3 regulated the transcription activity of CiBcl-2, finally led to a decrease of autophagy. In addition, when cells were in the state of autophagy, apoptosis was increased and cell viability was decreased. When CiSTAT3 was activated, cell apoptosis weakened and cell viability was increased. The results suggest that CiSTAT3 plays an important role in maintaining the normal physiological process of cells.


Assuntos
Autofagia/genética , Carpas/fisiologia , Proteínas de Peixes/genética , Regulação da Expressão Gênica/imunologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Fator de Transcrição STAT3/genética , Regulação para Cima/imunologia , Animais , Carpas/imunologia , Dimerização , Proteínas de Peixes/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fator de Transcrição STAT3/metabolismo
11.
J Exp Clin Cancer Res ; 38(1): 183, 2019 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-31053148

RESUMO

BACKGROUND: Acquired resistance to sorafenib greatly limits its therapeutic efficiency in the treatment of hepatocellular carcinoma (HCC). Increasing evidence indicates that long noncoding RNAs (lncRNAs) play important roles in the resistance to anti-cancer drugs. The present study aims to explore the involvement of lncRNA SNHG1 (small nucleolar RNA host gene 1) in sorafenib resistance and how SNHG1 is associated with overexpressed microRNA-21 (miR-21) and the activated Akt pathway, which have been demonstrated to mediate this resistance in HCC cells. METHODS: Sorafenib-resistant HCC (SR-HCC) cells were generated and their sorafenib-resistant properties were confirmed by cell viability and apoptosis assays. Potential lncRNAs were screened by using multiple bioinformatics analyses and databases. The expression of genes and proteins was detected by qRT-PCR, Western blot and in situ hybridization. Gene silencing was achieved by specific siRNA or lncRNA Smart Silencer. The effects of anti-SNHG1 were evaluated in vitro and in experimental animals by using quantitative measures of cell proliferation, apoptosis and autophagy. The binding sites of miR-21 and SNHG1 were predicted by using the RNAhybrid algorithm and their interaction was verified by luciferase assays. RESULTS: The Akt pathway was highly activated by overexpressed miR-21 in SR-HCC cells compared with parental HCC cells. Among ten screened candidates, SNHG1 showed the largest folds of alteration between SR-HCC and parental cells and between vehicle- and sorafenib-treated cells. Overexpressed SNHG1 contributes to sorafenib resistance by activating the Akt pathway via regulating SLC3A2. Depletion of SNHG1 enhanced the efficacy of sorafenib to induce apoptosis and autophagy of SR-HCC cells by inhibiting the activation of Akt pathway. Sorafenib induced translocation of miR-21 to the nucleus, where it promoted the expression of SNHG1, resulting in upregulation of SLC3A2, leading to the activation of Akt pathway. In contrast, SNHG1 was shown to have little effect on the expression of miR-21, which downregulated the expression of PTEN, leading to the activation of the Akt pathway independently of SNHG1. CONCLUSIONS: The present study has demonstrated that lncRNA SNHG1 contributes to sorafenib resistance by activating the Akt pathway and its nuclear expression is promoted by miR-21, whose nuclear translocation is induced by sorafenib. These results indicate that SNHG1 may represent a potentially valuable target for overcoming sorafenib resistance for HCC.


Assuntos
Carcinoma Hepatocelular/genética , Cadeia Pesada da Proteína-1 Reguladora de Fusão/genética , Neoplasias Hepáticas/genética , MicroRNAs/genética , RNA Longo não Codificante/genética , Animais , Apoptose/efeitos dos fármacos , Autofagia/genética , Carcinoma Hepatocelular/patologia , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Células Hep G2 , Humanos , Neoplasias Hepáticas/patologia , Camundongos , PTEN Fosfo-Hidrolase/genética , Proteínas Proto-Oncogênicas c-akt/genética , RNA Interferente Pequeno/genética , Sorafenibe/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
12.
J Exp Clin Cancer Res ; 38(1): 185, 2019 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-31053152

RESUMO

BACKGROUND: Malignant peripheral nerve sheath tumours (MPNSTs) are sarcomas of Schwann cell lineage origin that occur sporadically or in association with the inherited syndrome, neurofibromatosis type 1 (NF1). This study aimed to examine the function of High mobility group protein A2 (HMGA2) in NF1 MPNST progression and the underlying molecular mechanism. METHODS: Immunohistochemistry (IHC) was used to detect HMGA2 expression in MPNST and neurofibroma patient samples. Cell Cycle Kit-8 (CCK-8) and 5-ethynyl-20-deoxyuridine (EdU) assays, terminal deoxynucleotidyl transferase-mediated nick end labelling, and transmission electron microscopy were performed to reveal HMGA2 functions in NF1 MPNST cells in vitro and in vivo. Chromatin immunoprecipitation sequencing (ChIP-Seq) and RNA sequencing (RNA-Seq) were used to detect HMGA2-modulated genes regulating autophagy and growth in NF1 MPNSTs in vitro and in vivo. RESULTS: NF1 MPNST samples exhibit higher HMGA2 positivity rates (13/16) than sporadic MPNST (16/41) and neurofibroma (0/7) patient samples. High HMGA2 expression is correlated with poor prognosis. Neurofibromin 1 (NF1)-deficient MPNST cells display elevated HMGA2 expression. Functional experiments revealed that HMGA2 knockdown inhibits NF1 MPNST cell growth in vitro and in vivo. In addition to promoting cell cycle arrest and apoptosis, HMGA2 knockdown inhibits autophagy, favouring cell death. RNA-Seq and ChIP-Seq revealed that HMGA2 directly activates the Musashi-2 (MSI2) promoter region, and MSI2 overexpression reverses autophagy and growth in shHMGA2-transfected cells. MSI2 interacts with Beclin1, and Beclin1 blockade inhibits autophagy, thereby inhibiting cell proliferation. CONCLUSIONS: HMGA2 knockdown regulates autophagy via MSI2-Beclin1 interactions to inhibit NF1 MPNST growth, revealing potential therapeutic targets for these untreatable tumours.


Assuntos
Proteína HMGA2/genética , Neurofibromina 1/genética , Neurofibrossarcoma/genética , Proteínas de Ligação a RNA/genética , Adulto , Apoptose/genética , Autofagia/genética , Proteína Beclina-1/genética , Linhagem da Célula/genética , Proliferação de Células/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Proteína HMGA2/antagonistas & inibidores , Humanos , Masculino , Pessoa de Meia-Idade , Neurofibrossarcoma/patologia , Células de Schwann/metabolismo , Células de Schwann/patologia , Transdução de Sinais/genética
13.
Arch Virol ; 164(8): 2005-2013, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31102052

RESUMO

We previously found that genetic factors are associated with a familial predisposition for developing liver cirrhosis and hepatocellular carcinoma during chronic hepatitis B virus (HBV) infection. Autophagy has been shown to play a role in HBV replication and the course of disease. More than 190 host genes have been identified that modify the process of autophagy, but which of these genes are involved in chronicity of HBV infection and how this occurs remains unclear. Chronic hepatitis B (CHB) patients were recruited to investigate the expression of autophagy-modulating genes in peripheral blood mononuclear cells (PBMCs). mRNA prepared from PBMCs from members of two families with clustering HBV infection, including 11 CHB patients and nine healthy spouses, was hybridized to high-density oligonucleotide arrays. Immunoblot analysis was used to determine the level of autophagy. Of the 192 autophagy-modulating genes, 18 were found to be differently expressed. Of these, 11 displayed decreased expression in CHB patients, while seven displayed increased expression compared to those in healthy controls. Functional analysis showed that these genes are closely involved in initiation, nucleation, elongation of phagophores, formation of autophagosomes, transportation to lysosomes, and the process of degradation. Western blot analysis revealed inhibited autophagy in PBMCs based on decreased lipidation of LC3II. A differential expression profile of autophagy-modulating genes was observed, and decreased autophagy in PBMCs could be closely associated with chronicity of HBV infection, suggesting a novel strategy for the treatment of patients with chronic HBV infection.


Assuntos
Proteínas Relacionadas à Autofagia/genética , Autofagia/genética , Redes Reguladoras de Genes/genética , Hepatite B Crônica/genética , Leucócitos Mononucleares/fisiologia , Autofagossomos/fisiologia , Análise por Conglomerados , Feminino , Vírus da Hepatite B/patogenicidade , Hepatite B Crônica/virologia , Humanos , Lisossomos/fisiologia , Masculino , RNA Mensageiro/genética
14.
Oncol Rep ; 41(6): 3555-3564, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31002368

RESUMO

Neoplastic transformation is characterized by metabolic rewiring to sustain the elevated biosynthetic demands of highly proliferative cancer cells. To obtain the precursors for macromolecule biosynthesis, cancer cells avidly uptake and metabolize glucose and glutamine. Thus, targeting the availability or metabolism of these nutrients is an attractive anticancer therapeutic strategy. To improve our knowledge concerning how cancer cells respond to nutrient withdrawal, the response to glutamine and/or glucose starvation was studied in human in vitro transformed fibroblasts, deeply characterized at the cellular and molecular level. Concomitant starvation of both nutrients led to rapid loss of cellular adhesion (~16 h after starvation), followed by cell death. Deprivation of glucose alone had the same effect, although at a later time (~48 h after starvation), suggesting that glucose plays a key role in enabling cell attachment to the extracellular matrix. Glutamine deprivation did not induce rapid cell death, but caused a prolonged arrest of cellular proliferation; the cells started dying only 96 h after starvation. Before massive cell death occurred, the effects of all the starvation conditions were reversible. Autophagy activation was observed in cells incubated in the absence of glucose for more than 48 h, while autophagy was not detected under the other starvation conditions. Markers of apoptotic cell death, such as caspase 3, caspase 9 and poly(ADP­ribose) polymerase 1 (PARP­1) proteolytic fragments, were not observed under any growth condition. Glucose and/or glutamine deprivation caused very rapid PARP­1 activation, with marked PARP­1 (poly­ADP) ribosylation and protein (poly­ADP) ribosylation. This activation was not due to starvation­induced DNA double­strand breaks, which appeared at the late stages of deprivation, when most cells died. Collectively, these results highlight a broad range of consequences of glucose and glutamine starvation, which may be taken into account when nutrient availability is used as a target for anticancer therapies.


Assuntos
Proliferação de Células/genética , Transformação Celular Neoplásica/genética , Glucose/metabolismo , Glutamina/metabolismo , Apoptose/genética , Autofagia/genética , Caspases/genética , Caspases/metabolismo , Pontos de Checagem do Ciclo Celular/genética , Morte Celular/genética , Transformação Celular Neoplásica/metabolismo , Quebras de DNA de Cadeia Dupla , Fibroblastos/metabolismo , Fibroblastos/patologia , Glucose/genética , Glutamina/genética , Humanos , Terapia de Alvo Molecular , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Inanição/genética , Inanição/metabolismo
15.
J Exp Clin Cancer Res ; 38(1): 160, 2019 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-30987650

RESUMO

BACKGROUND: Glucose-6-phospate dehydrogenase (G6PD) is the limiting enzyme of the pentose phosphate pathway (PPP) correlated to cancer progression and drug resistance. We previously showed that G6PD inhibition leads to Endoplasmic Reticulum (ER) stress often associated to autophagy deregulation. The latter can be induced by target-based agents such as Lapatinib, an anti-HER2 tyrosine kinase inhibitor (TKI) largely used in breast cancer treatment. METHODS: Here we investigate whether G6PD inhibition causes autophagy alteration, which can potentiate Lapatinib effect on cancer cells. Immunofluorescence and flow cytometry for LC3B and lysosomes tracker were used to study autophagy in cells treated with lapatinib and/or G6PD inhibitors (polydatin). Immunoblots for LC3B and p62 were performed to confirm autophagy flux analyses together with puncta and colocalization studies. We generated a cell line overexpressing G6PD and performed synergism studies on cell growth inhibition induced by Lapatinib and Polydatin using the median effect by Chou-Talay. Synergism studies were additionally validated with apoptosis analysis by annexin V/PI staining in the presence or absence of autophagy blockers. RESULTS: We found that the inhibition of G6PD induced endoplasmic reticulum stress, which was responsible for the deregulation of autophagy flux. Indeed, G6PD blockade caused a consistent increase of autophagosomes formation independently from mTOR status. Cells engineered to overexpress G6PD became resilient to autophagy and resistant to lapatinib. On the other hand, G6PD inhibition synergistically increased lapatinib-induced cytotoxic effect on cancer cells, while autophagy blockade abolished this effect. Finally, in silico studies showed a significant correlation between G6PD expression and tumour relapse/resistance in patients. CONCLUSIONS: These results point out that autophagy and PPP are crucial players in TKI resistance, and highlight a peculiar vulnerability of breast cancer cells, where impairment of metabolic pathways and autophagy could be used to reinforce TKI efficacy in cancer treatment.


Assuntos
Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Glucosefosfato Desidrogenase/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/genética , Neoplasias da Mama/genética , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Sinergismo Farmacológico , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/genética , Feminino , Expressão Gênica , Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/metabolismo , Humanos , Lapatinib/farmacologia , Prognóstico , Recidiva
16.
J Exp Clin Cancer Res ; 38(1): 163, 2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30987661

RESUMO

BACKGROUND: ALKBH5 regulated the malignant behavior of breast cancer and glioblastoma. However, the expression and function of ALKBH5 in epithelial ovarian cancer have not yet been determined. In the present study, we investigated the expression and function of ALKBH5 in epithelial ovarian cancer with respect to its potential role in the tumorigenesis of the disease as well as an early diagnostic marker. METHODS: Immunohistochemistry and western blot were used to detect protein expression. Gene silencing and over-expression experiment were used to study gene function. Cell proliferation assay and Matrigel invasion assays were used to detect cell proliferation and invasion, respectively. The nude mouse tumor formation experiment was used to evaluate the growth of cells in vivo. RESULTS: The expression of ALKBH5 was found to be increased in epithelial ovarian cancer tissue as compared to the normal ovarian tissues. The silencing of ALKBH5 in SKOV3 cells enhanced the autophagy and inhibited the proliferation and invasion in vitro and in vivo, whereas the ectopic expression of ALKBH5 in A2780 cells exerted an opposite effect. Mechanical study revealed that ALKBH5 physically interacted with HuR. ALKBH5 activated EGFR-PIK3CA-AKT-mTOR signaling pathway. Also, ALKBH5 enhanced the stability of BCL-2 mRNA and promoted the interaction between Bcl-2 and Beclin1. CONCLUSION: Overall, the present study identified ALKBH5 as a candidate oncogene in epithelial ovarian cancer and a potential target for ovarian cancer therapy.


Assuntos
Homólogo AlkB 5 da RNA Desmetilase/genética , Autofagia/genética , Carcinoma Epitelial do Ovário/genética , Carcinoma Epitelial do Ovário/metabolismo , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Adulto , Idoso , Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Animais , Proteína Beclina-1/metabolismo , Carcinoma Epitelial do Ovário/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células , Metilação de DNA , Modelos Animais de Doenças , Receptores ErbB/metabolismo , Feminino , Xenoenxertos , Humanos , Imuno-Histoquímica , Camundongos , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Interferência de RNA
17.
Cell Biol Int ; 43(6): 582-592, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30958602

RESUMO

Cell death was once believed to be the result of one of two distinct processes, apoptosis (also known as programmed cell death) or necrosis (uncontrolled cell death); in recent years, however, several other forms of cell death have been discovered highlighting that a cell can die via a number of differing pathways. Apoptosis is characterised by a number of characteristic morphological changes in the structure of the cell, together with a number of enzyme-dependent biochemical processes. The result being the clearance of cells from the body, with minimal damage to surrounding tissues. Necrosis, however, is generally characterised to be the uncontrolled death of the cell, usually following a severe insult, resulting in spillage of the contents of the cell into surrounding tissues and subsequent damage thereof. Failure of apoptosis and the resultant accumulation of damaged cells in the body can result in various forms of cancer. An understanding of the pathways is therefore important in developing efficient chemotherapeutics. It has recently become clear that there exists a number of subtypes of apoptosis and that there is an overlap between apoptosis, necrosis and autophagy. The goal of this review is to provide a general overview of the current knowledge relating to the various forms of cell death, including apoptosis, necrosis, oncosis, pyroptosis and autophagy. This will provide researchers with a summary of the major forms of cell death and allow them to compare and contrast between them.


Assuntos
Morte Celular/genética , Morte Celular/fisiologia , Animais , Apoptose/genética , Apoptose/fisiologia , Autofagia/genética , Autofagia/fisiologia , Humanos , Necrose/genética , Necrose/metabolismo , Transdução de Sinais
18.
Mol Cancer ; 18(1): 82, 2019 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-30953511

RESUMO

BACKGROUND: Gallbladder cancer is the most common biliary tract malignancy and not sensitive to chemotherapy. Autophagy is an important factor prolonging the survival of cancer cells under chemotherapeutic stress. We aimed to investigate the role of long non-coding RNAs (lncRNAs) in autophagy and chemoresistance of gallbladder cancer cells. METHODS: We established doxorubicin (Dox)-resistant gallbladder cancer cells and used microarray analysis to compare the expression profiles of lncRNAs in Dox-resistant gallbladder cancer cells and their parental cells. Knockdown or exogenous expression of lncRNA combined with in vitro and in vivo assays were performed to prove the functional significance of lncRNA. The effects of lncRNA on autophagy were assessed by stubRFP-sensGFP-LC3 and western blot. We used RNA pull-down and mass spectrometry analysis to identify the target proteins of lncRNA. RESULTS: The drug-resistant property of gallbladder cancer cells is related to their enhanced autophagic activity. And we found a lncRNA ENST00000425894 termed gallbladder cancer drug resistance-associated lncRNA1 (GBCDRlnc1) that serves as a critical regulator in gallbladder cancer chemoresistance. Furthermore, we discovered that GBCDRlnc1 is upregulated in gallbladder cancer tissues. Knockdown of GBCDRlnc1, via inhibiting autophagy at initial stage, enhanced the sensitivity of Dox-resistant gallbladder cancer cells to Dox in vitro and in vivo. Mechanically, we identified that GBCDRlnc1 interacts with phosphoglycerate kinase 1 and inhibits its ubiquitination in Dox-resistant gallbladder cancer cells, which leads to the down-regulation of autophagy initiator ATG5-ATG12 conjugate. CONCLUSIONS: Our findings established that the chemoresistant driver GBCDRlnc1 might be a candidate therapeutic target for the treatment of advanced gallbladder cancer.


Assuntos
Autofagia/genética , Neoplasias da Vesícula Biliar/genética , Regulação Neoplásica da Expressão Gênica , Fosfoglicerato Quinase/genética , RNA Longo não Codificante/genética , Idoso , Animais , Antibióticos Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Proteína 12 Relacionada à Autofagia/genética , Proteína 12 Relacionada à Autofagia/metabolismo , Proteína 5 Relacionada à Autofagia/genética , Proteína 5 Relacionada à Autofagia/metabolismo , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Neoplasias da Vesícula Biliar/tratamento farmacológico , Neoplasias da Vesícula Biliar/metabolismo , Neoplasias da Vesícula Biliar/patologia , Humanos , Masculino , Camundongos , Camundongos Nus , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Pessoa de Meia-Idade , Oligorribonucleotídeos/genética , Oligorribonucleotídeos/metabolismo , Fosfoglicerato Quinase/metabolismo , RNA Longo não Codificante/agonistas , RNA Longo não Codificante/antagonistas & inibidores , RNA Longo não Codificante/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Ubiquitinação/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Mol Cancer ; 18(1): 85, 2019 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-30971271

RESUMO

BACKGROUND: Lung cancer patients with KRAS mutation(s) have a poor prognosis due in part to the development of resistance to currently available therapeutic interventions. Development of a new class of anticancer agents that directly targets KRAS may provide a more attractive option for the treatment of KRAS-mutant lung cancer. RESULTS: Here we identified a small molecule KRAS agonist, KRA-533, that binds the GTP/GDP-binding pocket of KRAS. In vitro GDP/GTP exchange assay reveals that KRA-533 activates KRAS by preventing the cleavage of GTP into GDP, leading to the accumulation of GTP-KRAS, an active form of KRAS. Treatment of human lung cancer cells with KRA-533 resulted in increased KRAS activity and suppression of cell growth. Lung cancer cell lines with KRAS mutation were relatively more sensitive to KRA-533 than cell lines without KRAS mutation. Mutating one of the hydrogen-bonds among the KRA-533 binding amino acids in KRAS (mutant K117A) resulted in failure of KRAS to bind KRA-533. KRA-533 had no effect on the activity of K117A mutant KRAS, suggesting that KRA-533 binding to K117 is required for KRA-533 to enhance KRAS activity. Intriguingly, KRA-533-mediated KRAS activation not only promoted apoptosis but also autophagic cell death. In mutant KRAS lung cancer xenografts and genetically engineered mutant KRAS-driven lung cancer models, KRA-533 suppressed malignant growth without significant toxicity to normal tissues. CONCLUSIONS: The development of this KRAS agonist as a new class of anticancer drug offers a potentially effective strategy for the treatment of lung cancer with KRAS mutation and/or mutant KRAS-driven lung cancer.


Assuntos
Antineoplásicos/farmacologia , Autofagia/genética , Benzoatos/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Proteínas Proto-Oncogênicas p21(ras)/química , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Antineoplásicos/química , Autofagia/efeitos dos fármacos , Benzoatos/química , Sítios de Ligação , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Guanosina Difosfato/química , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Camundongos Nus , Camundongos Transgênicos , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Proto-Oncogênicas p21(ras)/agonistas , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais , Bibliotecas de Moléculas Pequenas/química , Ensaios Antitumorais Modelo de Xenoenxerto
20.
Mol Cancer ; 18(1): 89, 2019 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-30999914

RESUMO

BACKGROUND: The biology function of antisense intronic long noncoding RNA (Ai-lncRNA) is still unknown. Meanwhile, cancer patients with paclitaxel resistance have limited therapeutic options in the clinic. However, the potential involvement of Ai-lncRNA in paclitaxel sensitivity remains unclear in human cancer. METHODS: Whole transcriptome sequencing of 33 breast specimens was performed to identify Ai-lncRNA EGOT. Next, the role of EGOT in regulation of paclitaxel sensitivity was investigated. Moreover, the mechanism of EGOT enhancing autophagy sensitizes paclitaxel cytotoxicity via upregulation of ITPR1 expression by RNA-RNA and RNA-protein interactions was investigated in detail. Furthermore, upstream transcriptional regulation of EGOT expression was also investigated by co-immunoprecipitation and chromatin immunoprecipitation. Finally, clinical breast specimens in our cohort, TCGA and ICGC were applied to validate the role of EGOT in enhancing of paclitaxel sensitivity. RESULTS: EGOT enhances autophagosome accumulation via the up-regulation of ITPR1 expression, thereby sensitizing cells to paclitaxel toxicity. Mechanistically, on one hand, EGOT upregulates ITPR1 levels via formation of a pre-ITPR1/EGOT dsRNA that induces pre-ITPR1 accumulation to increase ITPR1 protein expression in cis. On the other hand, EGOT recruits hnRNPH1 to enhance the alternative splicing of pre-ITPR1 in trans via two binding motifs in EGOT segment 2 (324-645 nucleotides) in exon 1. Moreover, EGOT is transcriptionally regulated by stress conditions. Finally, EGOT expression enhances paclitaxel sensitivity via assessment of cancer specimens. CONCLUSIONS: These findings broaden comprehensive understanding of the biology function of Ai-lncRNAs. Proper regulation of EGOT may be a novel synergistic strategy for enhancing paclitaxel sensitivity in cancer therapy.


Assuntos
Autofagia/genética , Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica , Receptores de Inositol 1,4,5-Trifosfato/genética , RNA Longo não Codificante/genética , Adulto , Idoso , Animais , Antineoplásicos Fitogênicos/farmacologia , Autofagossomos/metabolismo , Autofagia/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H/metabolismo , Humanos , Receptores de Inositol 1,4,5-Trifosfato/antagonistas & inibidores , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Paclitaxel/farmacologia , Ligação Proteica , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , RNA Longo não Codificante/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Análise de Sobrevida , Transcriptoma , Ensaios Antitumorais Modelo de Xenoenxerto
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