Presence Of Autophagy Research Articles

Reciprocal inhibition of autophagy and Botrytis cinerea-induced programmed cell death in ‘Shine Muscat’ grapes

Botrytis cinerea causes gray mold, decreasing the quality of table grapes. The berry response to B. cinerea infection was explored in present study, focusing on the relationship between presence of autophagy and programmed cell death (PCD). Results demonstrated B. cinerea infection decreased cell viability, triggering cell death, possibly resulting in PCD occurrence. It was further verified by increased terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive nuclei, heightened caspase 3-like and caspase 9-like protease activity, and elevated expression of metacaspase genes. Additionally, autophagy was indicated by the increased VvATG expression and autophagosome formation. Notably, the autophagy activator rapamycin reduced TUNEL-positive nuclei, whereas the autophagy inhibitor 3-methyladenine increased caspase 9-like protease activity. The PCD activator C2-ceramide inhibited autophagy, whereas the PCD inhibitor Acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO) enhanced autophagy gene expression. Autophagy and B. cinerea-induced PCD in berry cells are reciprocally negatively regulated; and the rapamycin and Ac-DEVD-CHO could potentially maintain table grape edible quality.

A tumor growth model with autophagy: The reaction-(cross-)diffusion system and its free boundary limit

<p style='text-indent:20px;'>In this paper, we propose a tumor growth model to incorporate and investigate the spatial effects of autophagy. The cells are classified into two phases: normal cells and autophagic cells, whose dynamics are also coupled with the nutrients. First, we construct a reaction-(cross-)diffusion system describing the evolution of cell densities, where the drift is determined by the negative gradient of the joint pressure, and the reaction terms manifest the unique mechanism of autophagy. Next, in the incompressible limit, such a cell density model naturally connects to a free boundary system, describing the geometric motion of the tumor region. Analyzing the free boundary model in a special case, we show that the ratio of the two phases of cells exponentially converges to a "well-mixed" limit. Within this "well-mixed" limit, we obtain an analytical solution of the free boundary system which indicates the exponential growth of the tumor size in the presence of autophagy in contrast to the linear growth without it. Numerical simulations are also provided to illustrate the analytical properties and to explore more scenarios.</p>

Autophagy and cellular senescence in classical Hodgkin lymphoma

Autophagy and cellular senescence are interrelated cellular stress responses important for cellular homeostasis and they have been implicated in the pathogenesis of classical Hodgkin lymphoma (cHL). However, the presence of autophagy and cellular senescence and their relation with clinical and laboratory parameters needs further elucidation. Thus, autophagy (LC3B and p62 immunohistochemical expression) and cellular senescence (p16 immunohistochemical expression and SenTraGor™ staining) were studied in tissue sections from 59 patients with cHL. Autophagy and cellular senescence-associated markers were detected in variable proportions of Hodgkin and Reed-Sternberg (HRS) cells in cHL cases. High nuclear p62 immunohistochemical expression in HRS cells showed significant positive correlation with relapse of the disease (p = 0.037). Heterogeneous autophagy and cellular senescence patterns were revealed in HRS cells suggesting biological heterogeneity of cHL. The detection of high nuclear p62 expression in HRS cells may identify a subset of cHL with more aggressive clinical behavior.

Prognostic value of gamma-interferon-inducible lysosomal thiol reductase expression in female patients diagnosed with breast cancer.

Because of the high heterogeneity of breast cancer outcome, identification of novel prognostic biomarkers is critical to improve patient stratification and guide precise treatment. We examined the prognostic value of gamma-interferon-inducible lysosomal thiol reductase (GILT) expression in a training set of 416 breast cancer patients and a validation set of 210 patients, and performed functional studies to investigate the functions and underlying mechanisms of GILT on breast cancer prognosis. Our results indicated that high GILT expression in breast cancer cells was associated with improved disease-free survival (DFS; hazard ratio [HR]=0.189, 95% confidence interval [CI]: 0.099-0.361) and breast cancer-specific survival (BCSS; HR=0.187, 95% CI: 0.080-0.437) of breast cancer patients both in the training set and the external validation set (HR=0.453, 95% CI: 0.235-0.873 for DFS, HR=0.488, 95% CI: 0.245-0.970 for BCSS). In vitro and in vivo studies showed that GILT overexpression inhibited breast cancer cells proliferation, invasion, migration and tumor formation in nude mice and increased sensitivity of breast cancer cells to standard treatment. Proteomics analysis indicated that GILT inhibited reactive oxygen species (ROS) and autophagy activation in breast cancer cells, and GILT overexpression-mediated tumor growth was further enhanced in the presence of autophagy or ROS inhibitors. Our results demonstrate that GILT expression can be effectively used to predict the prognosis and guide treatment strategies of breast cancer patients.

Alcoholic extract of Tarantula cubensis (Theranekron®) induce autophagy on gastric cancer cells

Aim: To evaluate the effects of theranekron in respect of autophagy on gastric cancer that is the fifth leading cancer type worldwide. Metods: In the present study, metastatic AGS and non-metastatic MKN-45 human gastric cell lines were used together with HEK-293 non-cancer cells as controls. Cytotoxic effect of theranekron besides appropriate treatment time was investigated through cell proliferation by using Cell Proliferation assay Kit (MTT) using different concentrations of the drug. The autophagic effect of the drug was determined using the LC3-GFP translocation assay and western blot analysis. All experiments were performed also using the ethanol since Tarantula cubensis spider was processed and diluted in 60% alcohol to generate as a drug. Results: MTT assay results demonstrated that the half maximal inhibitory concentration of theranekron was ~100 μM, its effect was found to be significant at 6 hrs, and theranekron decreased the cell viability in all cell lines without specificity in respect to the increasing concentrations. Additionally, a significantly increased GFP accumulation was detected in the autophagosomes of the cells treated with theranekron compared to non-treated cells, indicating the presence of autophagy. Conclusion: These findings were confirmed by LC3-I to LC3-II conversion with the western blot analysis. The data of ethanol experiments; however, demonstrated that ethanol also induced a cytotoxic effect and autophagic cell death. Our results suggested that theranekron results in cell death and stimulate autophagy process, but it is not specific for cancer cells since it represented similar results on non-cancer control cells. Moreover, the effect of theranekron on cell death might mostly occur through alcohol in which it is extracted.

Blocking Parkin/PINK1-mediated mitophagy sensitizes hepatocellular carcinoma cells to sanguinarine-induced mitochondrial apoptosis

Hepatocellular carcinoma (HCC) remains a major clinical challenge. Although mitophagy is implicated in hepatocarcinogenesis, novel therapeutic options targeting mitophagy for HCC treatment still await further studies. Here, we demonstrate that sanguinarine induces cell death in HCC cell line MHCC-97H through the mitochondrial apoptosis pathway. Sanguinarine triggers mitochondrial dysfunction and PTEN-induced putative kinase 1 (PINK1)/Parkin upregulation and recruitment to mitochondria. Elevated levels of p62 and LC3-II/I ratios suggest that sanguinarine is both an inducer of autophagy and a blocker of autolysosome formation, which is further confirmed by LC3-II conversion levels in presence of autophagy and mitophagy inhibitors, as well as an autophagy activator. In addition, blocking autophagy promotes sanguinarine-induced cell death, indicating mitophagy plays a cytoprotective role in sanguinarine-treated cells. Our findings suggest that blocking mitophagy may contribute to sanguinarine-induced mitochondrial apoptosis through the prevention of damaged mitochondrial clearance.

Combined Treatment of 5-Fluorouracil and Delta-Tocotrienol Induce of Apoptosis and Autophagy in Colorectal Cancer Cells

Background: 5-fluorouracil (5-FU) has been a first choice chemotherapeutic drug used to treat colorectal cancer. However, its therapeutic outcome often be limited by high-dose toxicities and drug resistance development. Hence, the current research advocates combined application of delta-tocotrienol (δ-T3), as a natural chemosensitizer, with 5-FU as a novel treatment approach for colorectal cancer. Aim: To investigate the combinatorial anticancer effects of δ-T3 and 5-FU on colorectal cancer cell lines. Methods: Cell viability assay was performed to investigate the individual and combined effects of δ-T3 (0.1-100 µM) and 5-FU (0.1-100 µM) on HCT116, HCC2998, Caco-2 and SW48 colorectal cancer cells at 72 h. Subsequently, the synergistic combination were evaluated for the effects on clonogenicity, apoptosis and autophagy. Morphologic assessment was conducted by bright-field and fluorescence microscopy. The protein expression profiles of programmed cell death and survival markers were determined by Western blotting. Results: Combined treatment of subeffective dose of δ-T3 significantly lowered the IC50 of 5-FU in Caco-2 and SW48 colorectal adenocarcinomas for 16-fold and fourfold respectively, signifying a chemosensitising effect. Clonogenic survival assay showed that the combined treatment profoundly hampered the cell survival as compared with the individual single treatments. Apoptosis was induced by the combined treatment as confirmed by flow cytometry. Interestingly, both apoptotic and autophagic morphologies were observed, including nuclear condensation, cell shrinkage, cytoplasmic vacuolation and extension. The presence of autophagy was further confirmed by increase LC3A/B ratio and high volume of acidic vesicular organelles. The combined treatment were found to upregulate prodeath proteins such as Bax, caspase-8, caspase-3, PARP and downregulate prosurvival proteins such as cIAP, XIAP and survivin. Conclusion: The enhanced cell death induced by 5-FU and δ-T3 combined treatment involved both apoptosis and autophagy, suggesting a novel and effective treatment approach for colorectal cancer.

Abstract 1261: Detection of autophagy in gastric mucosae of Chilean individuals residing in a geographic region characterized by high rates of gastric cancer-related death

Abstract Gastric cancer is the fourth most common cancer worldwide. In Chile, gastric cancer is the first cause of cancer-related deaths, affecting most prominently the southern provinces. Therefore, the elucidation of molecular pathways that might be exploited to improve prognosis and therapies remains an important challenge. There is emerging evidence pointing to the role of autophagy in tumorigenesis. Autophagy, a lysosome-mediated catabolic process, represents a prosurvival process implemented by neoplastic cells in response to stressful stimuli. So far, markers of autophagy have been used as prognosis determinants in early stages of gastric cancer, or have been associated to recurrence and reduced survival in patients with advanced disease. However, the role of autophagy in precursor lesions (chronic gastritis, metaplasia, dysplasia) has not been explored. Our goal was to examine the presence of autophagy (cleaved-LC3A) and proliferation (Ki-67) markers in gastric biopsies obtained from healthy individuals residing in a Chilean geographic zone characterized by a high gastric cancer-related mortality. Toward this goal, 165 asymptomatic individuals, aged 40 to 70, from the Molina area in the south of Chile were subjected to endoscopy and biopsy sampling according to the Operative Link on Gastritis Assessment (OLGA) staging system. Each tissue section was processed for immunohistochemical detection of cleaved-LC3A and Ki67, and the results were correlated with the histopathological findings and the presence or absence of Helicobacter pylori. Our preliminary analyses indicate that 60% of individuals harbor at least one out of five biopsies with the highest score values for cleaved-LC3A staining. Most of these high-scored samples involved the gastric antrum (73.3%) and were also positive for the Ki67 proliferation marker (63.3%). Of note, tissue samples showing the highest score values for cleaved-LC3A staining occurred almost always (93.3%) in the context of chronic gastritis or a combination of chronic gastritis and intestinal metaplasia, even though only 56.7% of cleaved-LC3A positive tissues showed signs of Helicobacter pylori infection. These results seem to indicate that autophagy may allow gastric epithelial cells to circumvent inflammatory stress, highlighting this process as a potential target for the development of anti-cancer or preventive therapies.Grant Support: FIC-30388034-0, CONICYT-FONDAP 115130011, Fondecyt 1151411, PIA-Gastric cancer UTALCA Citation Format: Alejandro Corvalan, Hernan Pulgar, Claudio Cruzat, Nicole Cespedes, Francisco Ruiz, Rodrigo Prado, Nelson E. Brown. Detection of autophagy in gastric mucosae of Chilean individuals residing in a geographic region characterized by high rates of gastric cancer-related death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1261.

Brd4 regulates the expression of essential autophagy genes and Keap1 in AML cells.

We have recently reported that activation of Brd4 is associated with the presence of autophagy in NPMc+ and MLL AML cells. In order to determine the mechanisms underlying this relationship, we have examined the role of Brd4 in regulating the expression of several genes that are central to the process of autophagy. We found that Brd4 binds to the promoters of ATG 3, 7 and CEBPβ, and expression of these genes is markedly reduced by inhibitors of Brd4, as well as by Brd4-shRNA and depletion of CEBPβ. Inhibitors of Brd4 also dramatically suppress the transcription of Keap1, thereby increasing the expression of anti-oxidant genes through the Nrf2 pathway and reducing the cytotoxicity induced by Brd4 inhibitors. Elimination of ATG3 or KEAP1 expression using CRISPR-cas9 mediated genomic editing markedly reduced autophagy. We conclude that Brd4 plays a significant role in autophagy activation through the direct transcriptional regulation of genes essential for it, as well as through the Keap1-Nrf2 axis in NPMc+ and MLL-fusion AML cells.

Ex vivo 3D human corneal stroma model for Schnyder corneal dystrophy - role of autophagy in its pathogenesis and resolution.

Multilamellar bodies (MLBs) are concentric cytoplasmic membranes which form through an autophagy-dependent mechanism. In the cornea, the presence of MLBs is associated with Schnyder corneal dystrophy (SCD). Ex vivo 3D modelling of the corneal stroma and SCD can help study pathogenesis and resolution of the disorder. Corneal stroma explants were isolated from cadavers and cultivated long-term for more than 3 months to achieve spontaneous 3D outgrowth of corneal stroma-derived mesenchymal stem-like cells (CSMSCs). The 3D tissues were then examined by transmission electron microscopy (TEM) for presence of MLBs, and by immunofluorescent labelling against markers for autophagy (p62, LC3). Autophagy was induced by classical serum starvation or rapamycin (RAP) treatment (50 nM), and inhibited by the autophagy inhibitor 3-methyladenine (3-MA, 10 mM) for 24 hours. CSMSCs can form spontaneously 3D outgrowths over a 3-4 weeks period, depositing their own extracellular matrix containing collagen I. TEM confirmed the presence of MLBs in the long-term (>3 months) 3D cultures, which became more abundant under starvation and RAP treatment, and decreased in number under autophagy inhibition with 3-MA. The presence of autophagy and its disappearance could be confirmed by an inversely related increase and decrease in the expression of LC3 and p62, respectively. MLB formation in long-standing CSMSC cultures could serve as a potential ex vivo model for studying corneal stroma diseases, including SCD. Inhibition of autophagy can decrease the formation of MLBs, which may lead to a novel treatment of the disease in the future.

The footprint of the ageing stroma in older patients with breast cancer

BackgroundTumours are not only composed of malignant cells but also consist of a stromal micro-environment, which has been shown to influence cancer cell behaviour. Because the ageing process induces accumulation of senescent cells in the body, this micro-environment is thought to be different in cancers occurring in old patients compared with younger patients. More specifically, senescence-related fibroblastic features, such as the senescence-associated secretory profile (SASP) and the induction of autophagy, are suspected to stimulate tumour growth and progression.MethodsWe compared gene expression profiles in stromal fields of breast carcinomas by performing laser capture microdissection of the cancer-associated stroma from eight old (aged ≥80 years at diagnosis) and nine young (aged <45 years at diagnosis) patients with triple-negative breast cancer. Gene expression data were obtained by microarray analysis (Affymetrix). Differential gene expression and gene set enrichment analysis (GSEA) were performed.ResultsDifferential gene expression analysis showed changes reminiscent of increased growth, de-differentiation and migration in stromal samples of older versus younger patients. GSEA confirmed the presence of a SASP, as well as the presence of autophagy in the stroma of older patients.ConclusionsWe provide the first evidence in humans that older age at diagnosis is associated with a different stromal micro-environment in breast cancers. The SASP and the presence of autophagy appear to be important age-induced stromal features.

Abstract P4-03-01: The footprint of the aging stroma in older breast cancer patients

Abstract Introduction: Tumoral masses are not only composed of malignant cells, but also enclose a more or less ample stromal micromilieu, which has been shown to influence the cancer cell behaviour. As aging induces accumulation of senescent cells in the body, this micromilieu is thought to be different in cancers occurring in old patients compared to the younger counterparts. More specifically, senescence-related fibroblastic features, such as the Senescence Associated Secretory Profile (SASP) and the induction of Autophagy, are suspected to stimulate tumor growth and progression. Materials and Methods: We compared gene expression profiles in stromal fields of breast carcinomas by performing laser capture microdissection of the cancer-associated stroma from 8 old (≥80 years at diagnosis) and 9 young (&amp;lt; 45 years at diagnosis) triple negative breast cancer patients. Gene expression data were obtained by microarray analysis (Affymetrix). Differential gene expression and Gene Set Enrichment Analysis (GSEA) were performed. Results: Differential gene expression analysis showed higher growth-, dedifferentiation- and migration- promoting gene expression in the stromal samples of older vs younger patients. GSEA confirmed the presence of a SASP, as well as the presence of Autophagy in the the stroma of older patients. Conclusion: We provide the first evidence in humans that older age at diagnosis is associated with a different stromal micromilieu in breast cancers. The SASP and the presence of Autophagy appear to be important age-induced stromal features.Introduction: Tumoral masses are not only composed of malignant cells, but also enclose a more or less ample stromal micromilieu, which has been shown to influence the cancer cell behaviour. As aging induces accumulation of senescent cells in the body, this micromilieu is thought to be different in cancers occurring in old patients compared to the younger counterparts. More specifically, senescence-related fibroblastic features, such as the Senescence Associated Secretory Profile (SASP) and the induction of Autophagy, are suspected to stimulate tumor growth and progression. Materials and Methods: We compared gene expression profiles in stromal fields of breast carcinomas by performing laser capture microdissection of the cancer-associated stroma from 8 old (≥80 years at diagnosis) and 9 young (&amp;lt; 45 years at diagnosis) triple negative breast cancer patients. Gene expression data were obtained by microarray analysis (Affymetrix). Differential gene expression and Gene Set Enrichment Analysis (GSEA) were performed. Results: Differential gene expression analysis showed higher growth-, dedifferentiation- and migration- promoting gene expression in the stromal samples of older vs younger patients. GSEA confirmed the presence of a SASP, as well as the presence of Autophagy in the the stroma of older patients. Conclusion: We provide the first evidence in humans that older age at diagnosis is associated with a different stromal micromilieu in breast cancers. The SASP and the presence of Autophagy appear to be important age-induced stromal features. Citation Format: Brouwers B, Fumagalli D, Brohee S, Hatse S, Govaere O, Floris G, Van den Eynde K, Schoffski P, Smeets A, Neven P, Lambrechts D, Sotiriou C, Wildiers H. The footprint of the aging stroma in older breast cancer patients [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-03-01.

Ultrastructure and Morphogenesis of Malsoor Virus: A Novel Phlebovirus Genetically Related to the Severe Fever with Thrombocytopenia Virus

The Malsoor virus (MV) is a novel Phlebovirus recently isolated from Rossetus bats from Western India that is closely related to Severe Fever and Thrombocytopenia virus (SFTS) and Heartland virus. Experimental studies showed pan cytotrophic property of the virus for different mammalian and insect cell lines in-vitro. High resolution transmission electron microscopy studies were carried out to characterize the fine structure and morphogenesis of this novel Phlebovirus. Detailed ultra structural studies on MV particles showed heterogeneous morphology. Majority of the virions (80%) were round having well defined envelope projections (6 ± 1nm) and having an average size of 70 nm. A subclass of virions (15%) showed pleomorphism. Interestingly, the envelope projections on the virions were tightly packed and not fragile to staining. Virus infected cells showed several interesting features not commonly reported for Bunya viruses. These included frequent detection of multiple inclusion bodies, distinct cytoplasmic membrane associated virus replication sites and presence of autophagy. This is the first electron microscopy study of this novel Phlebovirus. This is the first report on the detailed Ultra structure of this novel virus and forms the basis of further studies to explore the functional biology of this virus through more advanced approaches like correlative and cryo electron microscopy.

P.1.g.083 Effects of apocynin on retrograde memory dysfunction and glial response induced by transient global cerebral ischemia in rats

50mM for U251 and U87 cell lines and 100 mM for C6 cell line). When applied in its IC50 concentration, desloratadine increased production of ROS in U251 (1.4 times, compared to the control) as well as in other cell lines. It was also noted an increased caspase activity which implies to caspase-dependent apoptosis. Using double staining with Annexin V (An) and propidium iodide (PI), other apoptotic marker (extravasation of phosphatidyl serine) was noted after the tretment (33% of apoptotic cells in comparison to control that had 1.7%). Cell cycle analysis showed higher percent of apoptotic cells in sub G0 phase in C6 cell line (increased FL2 fluorescence). Increased presence of the acid cytoplasmic vesicles (quantified as an increase of the orange-FL3 vs. greenFL1 fluorescence, compared to the control) indicated a presence of autophagy in all investigated cell lines. Immunoblot method revealed the increased expression of LC3II after 18 h and 24 h of treatment, in dose-dependent manner. Conclusion: Desloratadine exerts cytotoxic effects on exammined cell lines, inducing apoptosis and autophagy. On view of the results showen above, we can conclude that its cytotoxic effect is not cell type speciffic.

Astin B, a cyclic pentapeptide from Aster tataricus, induces apoptosis and autophagy in human hepatic L-02 cells

Astins (including astin B) are a class of halogenated cyclic pentapeptides isolated from the medicinal herb of Aster tataricus. However, our previous works showed that the herbal medicine was hepatotoxic in vivo, and a toxicity-guided isolation method led to the identification of a cyclopeptide astin B. Astin B is structurally similar to cyclochlorotine, a well-known hepatotoxic mycotoxin. Thus, the aim of this study was to determine the potential cytotoxic effects and the underlying mechanism of astin B on human normal liver L-02 cells. We found that astin B has hepatotoxic effects in vitro and in vivo and that hepatic injury was primarily mediated by apoptosis in a mitochondria/caspase-dependent manner. Astin B provoked oxidative stress-associated inflammation in hepatocytes as evidenced by increased levels of reactive oxygen species (ROS), reduced contents of intracellular glutathione (GSH), and enhanced phosphorylation of c-Jun N-terminal kinase (JNK). Furthermore, the mitochondria-dependent apoptosis was evidenced by the depolarization of the mitochondrial membrane potential, the release of cytochrome c into cytosol, the increased ratio of Bax/Bcl-2, and the increased activities of caspases-9 and -3. Interestingly, astin B treatment also induces autophagy in L-02 cells, characterized by acidic-vesicle fluorescence, increased LC3-II and decreased p62 expression. Autophagy is a protective mechanism that is used to protect cells from apoptosis. The presence of autophagy is further supported by the increased cytotoxicity and the enhanced cleaved caspase-3 after co-treatment of cells with an autophagy inhibitor, also by increased LC3-II and decreased p62 after co-treatment with a caspase inhibitor. Taken together, astin B, most likely together with other members of astins, is the substance that is primarily responsible for the hepatotoxicity of A.tataricus.

Cloning of PaAtg8 and roles of autophagy in adaptation to starvation with respect to the fat body and midgut of the Americana cockroach, Periplaneta americana

Starvation, in particular amino acid deprivation, induces autophagy in trophocytes (adipocytes), the major component of the fat body cell types, in the larvae of Drosophila melanogaster. However, the fat body of cockroach has two additional cell types: urocytes depositing uric acid in urate vacuoles as a nitrogen resource and mycetocytes harboring an endosymbiont, Blattabacterium cuenoti, which can synthesize amino acids from the metabolites of the stored uric acid. These cells might complement the roles of autophagy in recycling amino acids in the fat body or other organs of cockroaches under starvation. We investigate the presence of autophagy in tissues such as the fat body and midgut of the American cockroach, Periplaneta americana, under starvation by immunoblotting with antibody against Atg8, a ubiquitin-like protein required for the formation of autophagosomes and by electron microscopy. Corresponding changes in acid phosphatase activity were also investigated as representing lysosome activity. Starvation increased the level of an autophagic marker, Atg8-II, in both the tissues, extensively stimulating the formation of autophagic compartments in trophocytes of the fat body and columnar cells of the midgut for over 2 weeks. Acid phosphatase showed no significant increase in the fat body of the starved cockroaches but was higher in the midgut of the continuously fed animals. Thus, a distinct autophagic mechanism operates in these tissues under starvation of 2 weeks and longer. The late induction of autophagy implies exhaustion of the stored uric acid in the fat body. High activity of acid phosphatase in the midgut of the fed cockroaches might represent enhanced assimilation and not an autophagy-related function.

Mitochondrial structure alteration in human prostate cancer cells upon initial interaction with a chemopreventive agent phenethyl isothiocyanate

BackgroundPhenethyl isothiocyanate (PEITC), present naturally in cruciferous vegetables, is a chemopreventive agent. It blocks initiation and post-initiation progression of carcinogenesis. Mechanism study in human prostate cancer cells revealed that PEITC is a dual inhibitor of aberrant DNA hypermethylation and histone deacetylases, reactivating silenced genes and regulating the androgen-mediated growth of tumor cells. The identity of the cellular organelle that initially interacts with PEITC has not been fully described.MethodsHuman prostate cancer LNCaP cells were exposed to PEITC and the effects on cellular fine structure examined by transmission electron microscopic studies. Alteration of mitochondrial membrane potential and cytochrome c release were evaluated as early events of apoptosis, and the TUNEL method for quantifying apoptotic cells. Mitochondria were isolated for determining their protein expression.ResultsUltrastructural analyses have revealed condensed mitochondria and a perturbed mitochondrial cristae structure, which assumed a rounded and dilated shape within 4-hours of PEITC contact, and became more pronounced with longer PEITC exposure. They presented as the most prominent intracellular alterations in the early hours. Mitochondria structure alterations were demonstrated, for the first time, with the isothiocyanates. An increase in the number of smooth endoplasmic reticulum and vacuoles were also noted that is consistent with the presence of autophagy. Early events of apoptosis were detected, with cytochrome c released along with the appearance of mitochondrial alteration. Mitochondrial membrane potential was disrupted within 18 hours of PEITC exposure, preceding the appearance of apoptotic cells with DNA strand breaks. In parallel, the expression of the mitochondrial class III ß-tubulin in the outer membrane, which associates with the permeability transition pore, was significantly reduced as examined with isolated mitochondria.ConclusionMitochondria may represent the organelle target of the isothiocyanates, indicating that the isothiocyanates may be mitochondria-interacting agents to inhibit carcinogenesis.

Genistein induces apoptosis and autophagy in human breast MCF-7 cells by modulating the expression of proapoptotic factors and oxidative stress enzymes

Breast cancer is one of the common tumors occurring in woman and despite treatment, the prognostic is poor. Genistein, a soy isoflavone, has been reported to have chemopreventive\chemotherapeutic potential in multiple tumor types. Here, we investigated the genistein antiproliferative effect in MCF-7 breast cancer, underlying the molecular mechanisms involved in this effect. MCF-7 cancer and CCD1059sK fibroblast cells were treated with estradiol (10 nM) or genistein (0.01-100 μM) for 24, 48, and 72 h and the cell proliferation was investigated by MTT; membrane cell permeability was evaluated by LDH and PI incorporation; apoptosis was investigated by externalization of phosphatidylserine by FACS; and presence of autophagy was detected by LC3A/B immunostaining. The expression of apoptotic proteins and antioxidant enzymes was evaluated by qPCR. The results demonstrate that genistein (100 μM) for 72 h of treatment selectively reduced MCF-7 cell proliferation independent of estrogen receptor activation, while no cytotoxicity was observed in fibroblast cells. Further experiments showed that genistein induced phosphatidylserine externalization and LC3A/B immunopositivity in MCF-7 cells, indicating apoptosis and autophagy cell death. Genistein increased in three times proapoptotic BAX/Bcl-2 ratio and promoted a parallel downregulation of 20 times of antiapoptotic survivin. In addition, genistein promoted a decrease of 5.5, 9.3, and 3.6 times of MnSOD, CuZnSOD, and TrxR mRNA expression, respectively, while the GPx expression was increased by 6.5 times. These results suggest that the antitumor effect of genistein involved the modulation of antioxidant enzyme and apoptotic signaling expression, which resulted in apoptosis and progression of autophagy.

The intersecting roles of endoplasmic reticulum stress, ubiquitin–proteasome system, and autophagy in the pathogenesis of proteinuric kidney disease

Protein misfolding in the endoplasmic reticulum (ER) leads to ER stress. The unfolded protein response and ER-associated degradation (ERAD) interact in a coordinated manner with the ubiquitin-proteasome system and autophagy to alleviate protein misfolding or its consequences. The intersecting actions of these processes are evident in normal podocyte physiology, and in proteinuric glomerular diseases, including experimental membranous nephropathy, focal segmental sclerosis, and diabetic nephropathy. There is some evidence for the induction of ER stress, changes in the ubiquitin-proteasome system, and presence of autophagy in human glomerulopathies. Various therapeutic approaches to the unfolded protein response, ERAD, and the ubiquitin-proteasome system have corrected experimental glomerular diseases involving protein misfolding, and could potentially be developed as therapies in humans.

Abstract 4113: Combination of LC3 II shRNA and genistein completely inhibited rapamycin-induced autophagy and promoted apoptosis in human malignant neuroblastoma cell lines

Abstract Neuroblastoma is an extracranial solid tumor that mainly occurs in children. It is derived from embryonic neural crest cells of the peripheral sympathetic nervous system. The overall survival rate of malignant neuroblastoma patients is very poor despite multi-modal therapy including surgery, radiotherapy, and chemotherapy. Efficacy of chemotherapy is often compromised due to presence of autophagy, which is a survival mechanism in solid tumors. Autophagy is a catablolic process for lysosomal degradation of cytoplasmic contents for recycling and it is activated during stress such as nutrient starvation and growth factor deprivation. The hallmark of autophagy is generation of double membrane structure called autophagosome that contains the microtubule-associated protein light chain 3 form II (LC3 II). In this study, we used rapamycin to mimic starvation-induced autophagy in human malignant neuroblastoma SK-N-BE2 and IMR32 cells and then investigated capability of the combination of LC3 II knockdown and genistein (GST) treatment for inhibition of autophagy and promotion of apoptosis. First, we treated both cell lines with 200 nM rapamycin for 6, 12, and 24 h and performed acridine orange (AO) staining, flow cytometric analysis, RT-PCR, and Western blotting to establish time-dependent increases in autophagic features such as formation of acidic vesicular organelles (AVO) and LC3 II. Then, we carried out dose-response studies by transfecting the rapamycin-exposed cell lines with various concentrations of LC3 II short hairpin RNA (shRNA) plasmid and treating them with several doses of GST alone and in combination. MTT assays showed the highest synergistic efficacy in inhibiting cell viability using the combination of 50 nM LC3 II shRNA and 25 µM GST in SK-N-BE2 cells while the combination of 100 nM LC3 II shRNA and 25 µM GST in IMR32 cells. Our AO staining and flow cytometric quantitation of AVO confirmed that combination of LC3 II shRNA and GST completely inhibited rapamycin-induced autophagy to promote apoptotic death. In situ Wright staining and Annexin V-FITC/PI staining showed that combination therapy most effectively induced apoptosis in both cell lines when compared with control shRNA, LC3 II shRNA, or GST alone. We unraveled the molecular mechanisms for inhibition of autopphagy and induction of apoptosis by Western blotting of the proteins involved in these processes. Down regulation of LC3 II and upregulation of mTOR in both cell lines showed inhibition of autophagy. Combination therapy promoted mitochondrial pathway leading to activation of caspase-3 and degradation of PARP for inducing apoptosis. Collectively, our results clearly show that combination of LC3 II knockdown and GST treatment serves as promising therapeutic strategy for inhibition of autophagy and induction of apoptosis in different human malignant neuroblastoma cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4113. doi:1538-7445.AM2012-4113