Autophagy-active beclin-1 correlates with favourable clinical outcome in non-Hodgkin lymphomas

Nicotra, Giuseppina; Mercalli, Francesca; Peracchio, Claudia; Castino, Roberta; Follo, Carlo; Valente, Guido; Isidoro, Ciro

doi:10.1038/modpathol.2010.80

Cited by 70 publications

(75 citation statements)

References 40 publications

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“…Beclin 1 is the mammalian ortholog of the yeast Vps30/Apg6 gene, which is required for autophagosome formation, and is monoallelically deleted in a high percentage of human carcinomas (28)(29)(30). Previous studies have revealed that the promotion of the expression of Beclin 1 through reduced autophagy demonstrates anticancer effects (25,26,31).…”

Section: Discussionmentioning

confidence: 99%

Role of autophagy in the ω-3 long chain polyunsaturated fatty acid-induced death of lung cancer A549 cells

Yao

Wang

et al. 2015

Oncology Letters

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Abstract. The present study identified that ω-3 long chain polyunsaturated fatty acids (ω-3 PUFAs), docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) demonstrate anti-proliferative effects in lung cancer A549 cells. MTS and cytotoxicity assays were conducted to confirm that ω-3 PUFAs induced cell death. Autophagy-associated gene and signaling pathways were also detected. Microtubule-associated protein light chain 3 (LC3) expression was found to be increased subsequent to treatment with DHA and EPA, and the expression of LC3-II was particularly increased. mRFP-GFP-LC3 fluorescence staining and p62 expression levels were used to detect autophagic flux. The present results indicate that DHA and EPA block autophagic flux, suggesting autophagosome accumulation. Subsequent to treatment with DHA and EPA, which interfered with autophagosomes, the expression of Beclin 1 was significantly decreased, while the expression of phosphorylated Akt and phosphorylated mammalian target of rapamycin was significantly increased. Therefore, DHA and EPA exert anti-proliferative effects by inhibiting autophagy in A549 cells, which highlights the potential of DHA and EPA for use in the prevention or treatment of lung cancer. IntroductionLung cancer is the leading cause of cancer-associated mortality worldwide (1), and the five-year survival rate remains extremely poor (2). Overall, ~75-85% of lung cancers cases are non-small cell lung cancer (NSCLC), which includes squamous cell carcinoma, adenocarcinoma and large cell carcinoma. The treatment of lung cancer involves the use of medical therapies such as surgery, radiation, chemotherapy and palliative care (3) in an attempt to successfully treat or reduce the adverse impact of malignant neoplasms originating in lung tissue. Chemotherapeutic agents are the main treatment measures for NSCLC, but the side-effects are usually difficult to tolerate (4).Fish oils are an excellent source of long-chain ω-3 polyunsaturated fatty acids (ω-3 PUFAs), including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Fish oil supplements are increasingly recognized by clinical studies to be useful for the treatment of a variety of human afflictions, including cancer (5). Numerous studies reveal evidence for the capability of ω-3 PUFAs to decrease proliferation, exert a pro-apoptotic effect and inhibit angiogenesis in several in vitro models of colon cancer (6-9).A previous study indicated that DHA and EPA inhibit the proliferation of A549 cells and induce apoptosis, with autophagy also being observed under transmission electron microscopy (10). Autophagy is a type of programmed cell death and it is an important process that is involved in various human pathologies. Previous studies suggest that autophagy is important in the regulation of cancer development and progression and also in determining the response of tumor cells to anticancer therapy (11)(12)(13)(14). Several cell signaling pathways are implicated in regulating autophagy, including the phosphatidyl inositol 3-kinase (PI3K)/Akt/...

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Section: Discussionmentioning

confidence: 99%

Role of autophagy in the ω-3 long chain polyunsaturated fatty acid-induced death of lung cancer A549 cells

Yao

Wang

et al. 2015

Oncology Letters

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“…139 When dealing with animal tissues, western blotting of LC3 should be performed on frozen biopsy samples homogenized in the presence of general protease inhibitors (Isidoro C, personal communication; see also Human). 140 Caveats regarding detection of LC3 by western blotting have been covered in a review. 25 For example, PVDF membranes may result in a stronger LC3-II retention than nitrocellulose membranes, possibly due to a higher affinity for hydrophobic proteins ( Fig.…”

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confidence: 99%

“…126 It is also possible to use anti-LC3/Atg8 antibodies for immunocytochemistry or immunohistochemistry, 140,[204][205][206][207][208][209] procedures that have the advantages of detecting the endogenous protein, obviating the need for transfection and or the generation of a transgenic organism, as well as avoiding potential artifacts resulting from overexpression (for example, high levels of overexpressed GFP-LC3 can result in its nuclear localization, although the protein can still relocate to the cytosol upon starvation), but we note that it is not always possible to detect endogenous Atg8/LC3. The use of imaging cytometry allows rapid and quantitative measures of the number of LC3 puncta and their relative number in individual or mixed cell types, using computerized assessment, enumeration, and data display (e.g., see refs.…”

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confidence: 99%

Guidelines for the use and interpretation of assays for monitoring autophagy

Klionsky¹,

Abdalla²,

Abeliovich³

et al. 2012

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In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

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“…21 Since BECN1 positivity is a hallmark of HBV-mediated autophagy, 8 we examined BECN1 in clinical liver tissues as previously described. 22,23 As shown in Fig. 6B, diffuse staining for BECN1 was observed in the cytoplasm of hepatocytes in healthy control tissues (regarded as negative), whereas large dark brown puncta were observed (regarded as positive) in those of CHB patients.…”

Section: Hbx Facilitates the Recruitment Of Tnfrsf10b To The Autophagmentioning

confidence: 99%

“…For analysis of BECN1 positivity, the levels of BECN1 macro-aggregates were assessed as described previously. 22 Representative areas were selected from scanned IHC images and cells were identified by hematoxylin staining of the nuclei. The percentage of cells with BECN1 macro-aggregates in the selected areas was determined.…”

Section: Immunohistochemical Analysis Of Liver Tissuesmentioning

confidence: 99%

Hepatitis B virus–triggered autophagy targets TNFRSF10B/death receptor 5 for degradation to limit TNFSF10/TRAIL response

et al. 2016

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Death receptors of TNFSF10/TRAIL (tumor necrosis factor superfamily member 10) contribute to immune surveillance against virus-infected or transformed cells by promoting apoptosis. Many viruses evade antiviral immunity by modulating TNFSF10 receptor signaling, leading to persistent infection. Here, we report that hepatitis B virus (HBV) X protein (HBx) restricts TNFSF10 receptor signaling via macroautophagy/autophagymediated degradation of TNFRSF10B/DR5, a TNFSF10 death receptor, and thus permits survival of virusinfected cells. We demonstrate that the expression of the TNFRSF10B protein is dramatically reduced both in liver tissues of chronic hepatitis B patients and in cell lines transfected with HBV or HBx. HBx-mediated downregulation of TNFRSF10B is caused by the lysosomal, but not proteasomal, degradation pathway. Immunoblotting analysis of LC3B and SQSTM1, and microscopy analysis of tandem-fluorescence-tagged LC3B revealed that HBx promotes complete autophagy. Inhibition of autophagy with a pharmacological inhibitor and LC3B knockdown revealed that HBx-induced autophagy is crucial for TNFRSF10B degradation. Immunoprecipitation and GST affinity isolation assays showed that HBx directly interacts with TNFRSF10B and recruits it to phagophores, the precursors to autophagosomes. We confirmed that autophagy activation is related to the downregulation of the TNFRSF10B protein in liver tissues of chronic hepatitis B patients. Inhibition of autophagy enhanced the susceptibility of HBx-infected hepatocytes to TNFSF10. These results identify the dual function of HBx in TNFRSF10B degradation: HBx plays a role as an autophagy receptor-like molecule, which promotes the association of TNFRSF10B with LC3B; HBx is also an autophagy inducer. Our data suggest a molecular mechanism for HBV evasion from TNFSF10-mediated antiviral immunity, which may contribute to chronic HBV infection.

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Autophagy-active beclin-1 correlates with favourable clinical outcome in non-Hodgkin lymphomas

Cited by 70 publications

References 40 publications

Role of autophagy in the ω-3 long chain polyunsaturated fatty acid-induced death of lung cancer A549 cells

Role of autophagy in the ω-3 long chain polyunsaturated fatty acid-induced death of lung cancer A549 cells

Guidelines for the use and interpretation of assays for monitoring autophagy

Hepatitis B virus–triggered autophagy targets TNFRSF10B/death receptor 5 for degradation to limit TNFSF10/TRAIL response

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