Degree Of Autophagy Research Articles

Halogenated Boroxine K2[B3O3F4OH] Modulates Metabolic Phenotype and Autophagy in Human Bladder Carcinoma 5637 Cell Line.

Halogenated boroxine K2[B3O3F4OH] (HB), an inorganic derivative of cyclic anhydride of boronic acid, is patented as a boron-containing compound with potential for the treatment of both benign and malignant skin changes. HB has effectively inhibited the growth of several carcinoma cell lines. Because of the growing interest in autophagy induction as a therapeutic approach in bladder carcinoma (BC), we aimed to assess the effects of HB on metabolic phenotype and autophagy levels in 5637 human bladder carcinoma cells (BC). Cytotoxicity was evaluated using the alamar blue assay, and the degree of autophagy was determined microscopically. Mitochondrial respiration and glycolysis were measured simultaneously. The relative expression of autophagy-related genes BECN1, P62, BCL-2, and DRAM1 was determined by real-time PCR. HB affected cell growth, while starvation significantly increased the level of autophagy in the positive control compared to the basal level of autophagy in the untreated negative control. In HB-treated cultures, the degree of autophagy was higher compared to the basal level, and metabolic phenotypes were altered; both glycolysis and oxidative phosphorylation (OXPHOS) were decreased by HB at 0.2 and 0.4 mg/mL. Gene expression was deregulated towards autophagy induction and expansion. In conclusion, HB disrupted the bioenergetic metabolism and reduced the intracellular survival potential of BC cells. Further molecular studies are needed to confirm these findings and investigate their applicative potential.

LncRNA FIRRE regulated endometrial cancer radiotherapy sensitivity via the miR-199b-5p/SIRT1/BECN1 axis-mediated autophagy

BackgroundEndometrial cancer (EC) poses a serious threat to women's health. Radiotherapy has been widely used for EC treatment. However, the mechanism of FIRRE in EC development and radioresistance remains unknown. MethodsMTT and colony formation assays determined cell proliferation. The degree of autophagy was tested by the measurement of autophagy-related genes and immunofluorescence staining of LC3. Molecular interactions were demonstrated via luciferase reporter assay, RIP, and Co-IP. The FIRRE role's was analyzed by in vivo xenograft tumor model. ResultsFIRRE and SIRT1 were upregulated in EC tumor tissues, whereas miR-199b-5p was reduced. FIRRE knockdown increased EC cell radiotherapy sensitivity by sponging miR-199b-5p and inhibiting autophagy. SIRT1 was targeted and negatively regulated by miR-199b-5p. SIRT1 could otherwise deacetylate BECN1 protein and participate in FIRRE-mediated autophagy. Silencing FIRRE increased sensitivity of EC radiotherapy in vivo. ConclusionFIRRE reduced EC cell radiotherapy sensitivity by stimulating autophagy via miR-199b-5p/SIRT1/BECN1 axis.

NLRP3 Inflammasome Simultaneously Involved in Autophagy and Phagocytosis of THP-1 Cells to Clear Aged Erythrocytes.

Autophagy and phagocytosis are two important processes that capture and digest materials found in cellular interiors and exteriors, respectively. Aged red blood cells (RBCs) are cleared by phagocytes in vivo. We focused on determining whether autophagy occurs after phagocytes swallow sunset erythrocytes, and whether the degree of autophagy is related to scavenging ability of phagocytes to erythrocytes. In addition, the ability of NLR family pyrin domain containing protein 3 (NLRP3) inflammasome to regulate erythrocyte clearance by phagocytes and its association with autophagy-related protein 16-like protein 1 (ATG16L1) are confirmed. We constructed a stable and low-NLRP3 expression THP-1 cell line using CRISPR/Cas9 technology. The analysis of erythrocyte clearance and autophagy of THP-1 cells with low NLRP3 expression showed that autophagy changes together when THP-1 engulfs aged RBCs. The occurrence of autophagy was dominated by microtubule-associated protein 1A/1B-light chain 3- (LC3-) associated phagocytosis accompanied by canonical autophagy. A negative correlation exists between the clearance of RBCs by THP-1 cells and the degree of autophagy. Downregulating the expression of NLRP3 in THP-1 cells can simultaneously inhibit the scavenging ability of THP-1 to erythrocytes and the degree of autophagy. In addition, the autophagy inhibitor bafilomycin A1 (BafA1) can enhance the phagocytosis ability of THP-1 to erythrocytes and promote the NLRP3 activation in THP-1 cells, while the autophagy inducer rapamycin inhibits the phagocytosis ability of THP-1 to RBCs and downregulates the NLRP3 activation. Results showed that autophagy and phagocytosis may be dynamic balance processes that can provide sufficient nutrition and energy to cells. Choosing NLRP3 as a target may regulate the phagocytic ability and the degree of autophagy in the meantime. These findings may be a potential strategy for regulating the clearance rate of phagocytes to aged RBCs and the secretion of proinflammatory cytokines to ensure transfusion safety.

Dynamic observation of 5-fluorouracil-induced myocardial injury and mitochondrial autophagy in aging rats.

Patients treated with 5-fluorouracil (5-FU) can develop rare but potentially severe cardiac effects, including cardiomyopathy, angina pectoris, heart failure and cardiogenic shock. The specific pathologies and underlying mechanisms are yet to be fully understood. The results of previous studies have indicated that mitochondrial autophagy is widely detected in many angiocardiopathies. In the present study, the dynamic changes in the homeostasis of mitochondrial injury and autophagy were observed in rats treated with 5-FU for different durations. A corresponding control group and a 5-FU model group were established in groups of Sprague-Dawley rats aged 2 and 18 months, and the myocardial enzyme levels were determined at different time points. At 2 weeks post-model establishment, cardiac ultrasound and myocardial histological staining were performed, cardiomyocyte apoptosis and myocardial mitochondrial function were assessed, and mitochondrial ultrastructure was examined. In addition, the expression levels of autophagy-related proteins were evaluated in the 18-month-old rats on days 7 and 14 of 5-FU administration. The experimental results demonstrated that 5-FU induced an elevation in the levels of myocardial enzymes, as well as changes in the cardiac structure and function, and that these changes were more prominent over longer drug durations. In addition, 5-FU decreased the levels of myocardial mitochondrial ATP and mitochondrial membrane potential, and aggravated myocardial fibrosis and cardiomyocyte apoptosis compared with those observed in the untreated control group, treated with the same volume of saline as 5-FU in the 5-FU group. These injuries were particularly evident in aging rats. Notably, 5-FU increased the expression levels of myocardial mitochondrial autophagy-related proteins, and electron microscopy revealed a more severe autophagic state in the model groups compared with that in the control groups. In conclusion, 5-FU induced myocardial mitochondrial damage, the degree of which was more severe in aging rats compared with that in young rats. The mitochondrial autophagy induced by 5-FU was excessive, and the degree of autophagy was aggravated with increased 5-FU administration time.

Vitexin Inhibits the Proliferation and Promotes the Apoptosis of Gastric Cancer Cells via Phosphatidylinositol-3-Kinase (PI3K)/Protein Kinase B (Akt)/The Mammalian Target of Rapamycin (mTOR) Signaling Pathway

Gastric cancer (GC) is a kind of digestive tract malignancy that has very high morbidity and mortality, making it crucial to find new drug treatments. Vitexin is a kind of flavonoid compound, which has anti-tumor, anti-inflammatory, analgesic and antiviral effects, etc. However, the specific role of vitexin in GC is still unclear. In this study, the expression of the survival rate and apoptosis was detected by CCK-8 and flow cytometry after vitexin acted on cells. Plasmid transfection technique was used to overexpress PI3K. Expression of PI3K/AKT/mTOR pathway-related proteins, autophagic-related proteins (Atg14, beclin-1, P62) and apoptotic-related proteins (bcl-2, Bax, cleaved caspase3) were detected by Western blot. We found that the cell survival rate decreased with the increasing time and dosage of vitexin. When vitexin acted on cells, the expression of p-PI3K, p-AKT and p-mTOR was significantly decreased, the degree of autophagy was increased, and the apoptosis rate was obviously increased. However, the overexpression of PI3K, the level of autophagy and apoptosis rate of cells which were given vitexin significantly decreased. In conclusion, Vitexin induces autophagy by inhibiting PI3K/AKT/mTOR signaling pathway, thereby inhibiting proliferation and promoting apoptosis of GC cells.

Autophagy in non-neoplastic skin diseases

ABSTRACT Background: Autophagy is an essential cellular mechanism that plays the housekeeping role in normal physiological processes, including the removal of long-lived, harvested, and misfolded proteins, damaged organs. On the other hand, under conditions causing cellular stress, such as malnutrition, hypoxia, oxidative stress, pathogen infections, radiation or cancer medication, there is an increase in the degree of autophagy, which leads to adaptation and cell survival. With the current interest in autophagy, this process appears to affect every organ and modify growing list of disease processes. The skin acts as the first line of defense against different environmental stresses; However, only a few studies have investigated the effect of autophagy on the pathogenesis of skin diseases. The level of autophagy may reflect the degree of some diseases; Therefore, detecting the level of autophagy may be an indirect means to assess some diseases. Objective: to provides an overview of recent findings relevant to the role of autophagy in non-neoplastic skin diseases. Conclusion: Autophagy plays an important role in mitigating or exacerbating various skin diseases. Understanding the mechanisms of autophagy and their regulation in different tissues and cells under healthy and stressful conditions will help better understand the etiology of skin diseases and develop more effective therapeutic approaches.

Chloroquine Downregulation of Intestinal Autophagy to Alleviate Biological Stress in Early-Weaned Piglets.

Simple SummaryWeaning is one of the biggest challenges in a pig’s life. Autophagy is a catabolic process aimed at recycling cellular components and damaged organelles in response to diverse stress conditions. There are two autophagy-modifying agents, rapamycin (RAPA) and chloroquine (CQ), that are often used in vitro and in vivo to regulate this process. We speculated that the regulation of autophagy may have some effect on weaning pressure. In this study, we try to understand the role of autophagy in intestinal barrier function and inflammation during the first week after weaning. We examined the effects of modulation of autophagy via RAPA and CQ on growth performance, immunity, inflammation profile, and the intestinal barrier to find potential value for CQ as a feed additive agent for ameliorating weaning stress.Early weaning stress impairs the development of gastrointestinal barrier function, causing immune system dysfunctions, reduction in feed intake, and growth retardation. Autophagy was hypothesized to be a key underlying cellular process in these dysfunctions. We conjectured that rapamycin (RAPA) and chloroquine (CQ), as two autophagy-modifying agents, regulate the autophagy process and may produce deleterious or beneficial effects on intestinal health and growth. To explore the effect of autophagy on early weaning stress in piglets, 18 early-weaned piglets were assigned to three treatments (each treatment of six piglets) and treated with an equal volume of RAPA, CQ, or saline. The degree of autophagy and serum concentrations of immunoglobulins and cytokines, as well as intestinal morphology and tight junction protein expression, were evaluated. Compared with the control treatment, RAPA-treated piglets exhibited activated autophagy and had decreased final body weight (BW) and average daily gain (ADG) (p < 0.05), impaired intestinal morphology and tight junction function, and higher inflammatory responses. The CQ-treated piglets showed higher final BW, ADG, jejuna and ileal villus height, and lower autophagy and inflammation, compared with control piglets (p < 0.05). Throughout the experiment, CQ treatment was beneficial to alleviate early weaning stress and intestinal and immune system dysfunction.

Regulating effect of electroacupuncture on left ventricular function and autophagy-specific proteins in spontaneously hypertension rats

To investigate the effect of electroacupuncture (EA) on cardiac hypertrophy and expression of myocardial autophagy-specific proteins in spontaneously hypertensive rats (SHRs). Twelve male Wistar Kyoto (WKY) rats were selected as the normal control group, and 24 male SHRs were randomized into model and EA groups (n＝12 in each). EA (2 Hz /15 Hz, 1 mA) was applied to "Taichong" (LR3) and "Baihui" (GV20) for 20 min, once a day for successive 30 days. The other two groups only received the same grasping and fixation procedures. The systolic blood pressure (SBP) was measured using a non-invasive blood detector, and the left ventricular function including the left ventricular anterior wall diameter (LVAWd), left ventricular posterior wall diameter (LVPWd) and left ventricular internal diameter at end-diastolic dimension (LVIDd) was detected using a real-time echocardiography imaging system, and the left ventricular mass index (left ventricle weight / body weight, LVMI) was calculated. The expression of Beclin-1 and LC3 proteins in the left ventricle tissue was detected by Western blot. Pathological changes of the myocardial tissue were observed by Masson staining and transmission electron microscope (TEM). After the intervention, the elevated SBP was significantly lowered in the EA group relevant to the model group (P<0.01) and the increased LVAWd, LVPWd and LVMI were markedly decreased in the EA group (P<0.01, P<0.05), while the reduced LVIDd was significantly ameliorated in the EA group (P<0.01). Masson staining showed that in SHRs, disorder of arrangement of cardiac myofibers, swelling of cardiomyocytes, widened space among myocardial cells with a large number of collagenous fibers were observed, and under TEM, aggregation and unequal size of myocardial mitochondria with vacuolation and rupture, autophagosomes and autophagic lysosomes were seen. These changes were relatively milder in the EA group. Western blot showed that hypertension induced up-regulation of Beclin-1 and LC3-II and ratio of LC3-II/LC3-I, and down-regulation of LC3-I were significantly suppressed in the EA group (P<0.01, P<0.05). EA at LR3 and GV20 may improve the left cardiac function, myocardial morphological changes and the degree of autophagy in SHRs, which is related to its effects in down-regulating the expression of myocardial Beclin-1 and LC3-II and in up-regulating expression of LC3-I.

Role of autophagy in a model of obesity: A long‑term high fat diet induces cardiac dysfunction.

Obesity may induce end-organ damage through metabolic syndrome, and autophagy serves a vital role in the pathogenesis of metabolic syndrome. The purpose of the present study was to define the roles of autophagy and mitophagy in high fat diet (HFD)-induced cardiomyopathy. Male, 8 week-old C57BL/6 mice were fed either a HFD (60% kcal) or a diet of normal chow (NC; 10% kcal) for 42 weeks. Glucose tolerance tests were performed during the feeding regimes. Blood samples were collected for assaying serum triglyceride with the glycerol-3-phosphate oxidase phenol and aminophenazone (PAP) method and total cholesterol was tested with the cholesterol oxidase-PAP method. Myocardial function was assessed using echocardiography and hemodynamic analyses. Western blot analysis was employed to evaluate endoplasmic reticulum stress (ERS), autophagy and mitochondrial function. Electron microscopy was used to assess the number of lipid droplets and the degree of autophagy within the myocardium. The body weight and adipose tissue weight of mice fed the HFD were increased compared with the NC mice. The serum levels of blood glucose, total cholesterol and triglyceride were significantly increased following 42 weeks of HFD feeding. The results of the glucose tolerance tests additionally demonstrated metabolic dysregulation in HFD mice. In addition, HFD mice exhibited hemodynamic and echocardiographic evidence of impaired diastolic and systolic function, including alterations in the cardiac output, end-diastolic pressure, end-diastolic volume and left ventricular relaxation time constant (tau) following HFD intake. Furthermore, a HFD resulted in increased ERS, and a downregulation of the autophagy and mitophagy level. The present study investigated cardiac function in obese HFD-fed mice. These results aid the pursuit of novel therapeutic targets to combat obesity-associated cardiomyopathy.

The alteration of autophagy and apoptosis in the hippocampus of rats with natural aging-dependent cognitive deficits

AimsThe present study was aim to explore aging-dependent changes in hippocampal autophagy and apoptosis in a natural aging rat model from adult to old stages and to discover a suitable age for treating neurodegenerative diseases. MethodsWistar rats at 5, 18 and 24months of age were used to mimic the adulthood, initial old, and old phases, respectively. The learning and cognitive ability of the rats was detected by the Morris water maze test. Morphological changes in the hippocampus were observed. Expressions of apoptosis and autophagy-related proteins were examined by Western blot. ResultsThe adult group (5months) exhibited high levels of autophagy related p-ULK p-ULK-1/ULK-1 ratio, Beclin-1, LC3II and cell survival, maintaining normal learning and cognitive function and integrated hippocampal morphology. The initial old group (18 months) presented a reduced number of neurons and cognitive deficits, and exhibited high levels of apoptosis related Bax/Bcl-2 ratio, Caspase-3 activation and autophagy related p-ULK p-ULK-1/ULK-1 ratio, Beclin-1, LC3II compared to the adult group. The old group (24 months) exhibited a high level of apoptosis related Bax/Bcl-2 ratio, Caspase-3 activation and a low level of autophagy related p-ULK p-ULK-1/ULK-1 ratio, Beclin-1, LC3II compared to its younger group, as well as significant neuronal death and cognitive deficits. The degree of autophagy was generally consistent with its negative regulator, the PI3K/Akt/mTOR axis, in all groups. ConclusionOur data suggest that cognitive deficits are first observed in the initial old stage. The levels of autophagy and apoptosis tend to be opposite in the adult and old phases. High levels of autophagy and apoptosis coexist in the initial old stage. Our study indicates that up-regulation of autophagy in the initial old phase to anti-cognitive deficits must be further evaluated.

Redox homeostasis protects mitochondria through accelerating ROS conversion to enhance hypoxia resistance in cancer cells.

Mitochondria are the powerhouses of eukaryotic cells and the main source of reactive oxygen species (ROS) in hypoxic cells, participating in regulating redox homeostasis. The mechanism of tumor hypoxia tolerance, especially the role of mitochondria in tumor hypoxia resistance remains largely unknown. This study aimed to explore the role of mitochondria in tumor hypoxia resistance. We observed that glycolysis in hypoxic cancer cells was up-regulated more rapidly, with far lesser attenuation in aerobic oxidation, thus contributing to a more stable ATP/ADP ratio. In hypoxia, cancer cells rapidly convert hypoxia-induced O2·− into H2O2. H2O2 is further decomposed by a relatively stronger antioxidant system, causing ROS levels to increase lesser compared to normal cells. The moderate ROS leads to an appropriate degree of autophagy, eliminating the damaged mitochondria and offering nutrients to promote mitochondria fusion, thus protects mitochondria and improves hypoxia tolerance in cancer. The functional mitochondria could enable tumor cells to flexibly switch between glycolysis and oxidative phosphorylation to meet the different physiological requirements during the hypoxia/re-oxygenation cycling of tumor growth.

N-n-butyl haloperidol iodide protects cardiomyocytes against hypoxia/reoxygenation injury by inhibiting autophagy.

N-n-butyl haloperidol iodide (F2), a novel compound derived from haloperidol, protects against the damaging effects of ischemia/reperfusion (I/R) injury in vitro and in vivo. In this study, we hypothesized the myocardial protection of F2 on cardiomyocyte hypoxia/reoxygenation (H/R) injury is mediated by inhibiting autophagy in H9c2 cells. The degree of autophagy by treatment with F2 exposed to H/R in H9c2 cell was characterized by monodansylcadaverine, transmission electron microscopy, and expression of autophagy marker protein LC3. Our results indicated that treatment with F2 inhibited autophagy in H9c2 cells exposed to H/R. 3-methyladenine, an inhibitor of autophagy, suppressed H/R-induced autophagy, and decreased apoptosis, whereas rapamycin, a classical autophagy sensitizer, increased autophagy and apoptosis. Mechanistically, macrophage migration inhibitory factor (MIF) was inhibited by F2 treatment after H/R. Accordingly, small interfering RNA (siRNA)-mediated MIF knockdown decreased H/R-induced autophagy. In summary, F2 protects cardiomyocytes during H/R injury through suppressing autophagy activation. Our results provide a new mechanistic insight into a functional role of F2 against H/R-induced cardiomyocyte injury and death.

Abstract 2899: Mitophagy imparts enzalutamide resistance in prostate cancer

Abstract Purpose: Anti-androgens are widely used in androgen deprivation therapy (ADT), a standard-of-care for patients with recurrent prostate cancer (PCa). Unfortunately, most patients ultimately develop resistance to ADT and progress to castrate-resistant prostate cancer (CRPC). Recently, two agents that block androgen receptor activation [abiraterone acetate (Zytiga, J&amp;J) and enzalutamide (Xtandi, Medivation/Astellas)] have been approved for CRPC therapy. While some patients respond to these therapies, many fail. We investigated if sequestration of metabolically aberrant mitochondria in the autophagosomes (mitophagy) imparts anti-androgen resistance. Method: We studied the effects of anti-androgen enzalutamide on the autophagy of androgen-dependent LNCaP and -independent C4-2 cells. Autophagy was monitored by cellular fluorescence in cells treated with monodansylcadavarine (MDC). Cellular fluorescence due to Mitosox dye oxidation was used to identify mitochondria producing high superoxide (O2-). Mitophagy of O2- producing mitochondria was monitored using fluorescence resonance energy transfer (FRET) between MDC and Mitosox in a 96-well plate based high throughput (HTS) assay and by visualization of FRET images and quantitation of FRET image intensities using a Nikon A1 fluorescence confocal microscope and associated software. Results: Our data show that in low androgen media, the degree of autophagy is less in C4-2 cells than in LNCaP cells. Enzalutamide treatment induces autophagy in both cell lines, but the increase in autophagy is more pronounced in the androgen-independent C4-2 than in the -dependent LNCaP cells. FRET data from both HTS and fluorescence microscopy show that while enzalutamide increases mitophagy of O2- producing mitochondria in C4-2 cells in a dose dependent manner, it decreases after an initial increase in LNCaP cells. Mitophagy of such mitochondria has also been observed by FRET based fluorescence microscopy in live circulating tumor cells (CTCs) isolated from blood sample of a patient undergoing enzalutamide therapy. Conclusion: Our data demonstrate that PCa cells resistant to enzalutamide show high degree of mitophagy of O2- mitochondria in autophagosomes. If this effect correlates with CTCs from blood collected from CRPC patients undergoing enzalutamide therapy, it can become a clinically useful method of identifying patients who are most likely to benefit from enzalutamide treatment. Citation Format: Hirak S. Basu, Cynthia L. Schrieber, Jamie M. Sperger, Maryanne Naundorf, Ashley M. Weichman, Farideh Mehraein-Ghomi, Dawn R. Church, Joshua M. Lang, George Wilding. Mitophagy imparts enzalutamide resistance in prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2899. doi:10.1158/1538-7445.AM2015-2899

Effects of 5‑fluorouracil and class III phosphoinositide 3‑kinase small interfering RNA combination therapy on SGC7901 human gastric cancer cells.

The aim of the present study was to investigate the effects of small interfering RNA‑mediated inhibition of Class III phosphoinositide 3‑kinase (PI3K) signal transduction on the proliferation, apoptosis and autophagy of SGC7901 gastric cancer cells. The present study also aimed to examine the contribution of autophagic inhibition to the antitumor effects of 5‑fluorouracil (5‑FU). A PI3K(III)‑RNA interference (i)‑green fluorescent protein (GFP) recombinant replication adenovirus (AD) and the negative control (NC)‑RNAi‑GFP control AD were constructed and infected into SGC7901 cells. A methyl thiazolyl tetrazolium assay was used to determine the growth rate of the SGC7901 cells. Immunofluorescent staining was used to detect microtubule‑associated protein 1 light chain 3 expression. The mitochondrial membrane potential was measured using the JC‑1 fluorescent probe. Autophagic expression was monitored with MDC staining and transmission electron microscopy. The results revealed that following combination treatment of the SGC7901 gastric cancer cells with 5‑FU + PI3K(III)‑RNAi‑AD, the optical density absorbance values at 24, 48 and 72 h were 0.17 ± 1.64, 0.13 ± 4.64 and 0.11 ± 3.56%, respectively, with cell viability inhibition ratios of 45.89 ± 6.67, 72.57 ± 9.48 and 87.51 ± 4.65%, respectively. As compared with the other treatment groups, the inhibition rate in the combined treatment group was significantly higher (P<0.05). The percentages of the cells with green fluorescence in the combined treatment group were 74.4 ± 3.86 (24 h), 82.3 ± 1.84 (48 h) and 92.5 ± 1.1% (72 h), which were larger than those of the other groups. The percentage of cells with green fluorescence became larger, which indicated that the mitochondrion membrane potential had been reduced to a greater extent. MDC staining revealed that the number of autophagic vacuoles in the cells (measured at 24, 48 and 72 h) decreased gradually with time, with more autophagic vacuoles observed in the cells in the control group at 24 h than those in the other treatment groups. Fewest autophagic vacuoles were identified in the combined treatment group. Using a fluorescence microscope, the immune fluorescence expression of microtubule‑associated proteins 1A/1B light chain 3A, which is the specific protein of autophagy, in the combined treatment group was observed to be significantly downregulated, as compared with the other groups. As determined by transmission electron microscopic observation of the SGC7901 gastric cancer cells, the degree of autophagy in the combined treatment group was significantly reduced, as compared with that of the other treatment groups. In conclusion, following combined treatment with 5‑FU and an inhibitor of class III PI3K signal transduction, the proliferation of SGC7901 cells was significantly suppressed, the mitochondrion membrane potentials were significantly reduced and the expression levels of autophagic markers were significantly downregulated.

Enhanced autophagy follows LMP1 expression disruption in EBV+ P3HR1 cells treated with arsenic trioxide.

Event Abstract Back to Event Enhanced autophagy follows LMP1 expression disruption in EBV+ P3HR1 cells treated with arsenic trioxide. Abderezak Zebboudj1*, Chafia Touil-Boukoffa1 and Mehdi Bourouba1 1 University of Sciences and Technologies Houari Boumediene (USTHB), FSB, Algeria Introduction : The Epstein Barr Virus (EBV) latency is associated with several malignancies among which nasopharyngeal carcinoma (NPC) and immunocompromised patients lymphoma. Expression and signaling of the major viral oncogene latent membrane protein 1 (LMP1) is essential to the immortalization of NPC cells and in vitro of B lymphocytes. Arsenic trioxide (ATO) treatment was reported to induce apoptosis in NPC cells via LMP1 down-regulation. Conversely, autophagy has been linked to LMP1 expression in B cells and survival. Here, we analyzed the anti-proliferative activity of ATO on P3HR1 EBV+ lymphoblastoid cells and assessed it's effect on apoptosis, autophagy and LMP1 expression. Materials and Methods : LMP1 expressing cells P3HR1 were treated with different concentrations of ATO. Apoptosis was measured by annexin V/PI double staining. DiOC6 labeling was assessed for mitochondrial potential (ΔΨm). Degree of autophagy was assessed based on LC3BI/LC3BII conversion. The anti-proliferative effect of ATO was studied using soft agar clonogenicity test. LMP1 expression and transcription was analyzed by western blot and Q-RT-PCR. Results : Our results show that ATO treatment leads to increased annexin V labeling and loss of mitochondrial potential in P3HR1 cells. We found that apoptosis is accompanied with enhanced autophagic activity as indicated by LC3B profile. Our results also indicate that ATO treatment induces disruption of LMP1 expression after 72H of treatment and inhibition of its transcription. Thus effects were associated with suppression of cells clonogenicity. Conclusion : Our study suggests that ATO cytotoxicity in P3HR1 cells occurs via induction of apoptosis and up-regulation of autophagic activity. These processes may occur as a consequence of LMP1 transcription repression and depletion of cellular LMP1 pools. Taken together, our data suggest that ATO may regulate negatively tumor cells survival by suppressing LMP1 anti-apoptotic signaling and enhancing autophagic cell death. Acknowledgements This work was performed and co-directed in collaboration with Dr Mounira Chelbi- Alix and Dr Sébastien Nisole team "INSERM UMR-S 1124, Université Paris Descartes,Paris, France". We thank Pr Mohamed Seghier "Institut Pasteur d'Algérie" for material and technical support. Keywords: ATO, apoptosis and autophagy, LMP1, ebv, Lymphoma, B-Cell Conference: The First International Congress of Immunology and Molecular Immunopathology (CIMIP2014), Tlemcen, Algeria, 17 Oct - 20 Oct, 2014. Presentation Type: Oral Poster Presentation Topic: Lymphoproliferative disorders Citation: Zebboudj A, Touil-Boukoffa C and Bourouba M (2014). Enhanced autophagy follows LMP1 expression disruption in EBV+ P3HR1 cells treated with arsenic trioxide.. Front. Immunol. Conference Abstract: The First International Congress of Immunology and Molecular Immunopathology (CIMIP2014). doi: 10.3389/conf.fimmu.2014.04.00007 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 10 Sep 2014; Published Online: 01 Dec 2014. * Correspondence: Mr. Abderezak Zebboudj, University of Sciences and Technologies Houari Boumediene (USTHB), FSB, Bab-Ezzouar, Algiers, 16111, Algeria, azebboudj@usthb.dz Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Abderezak Zebboudj Chafia Touil-Boukoffa Mehdi Bourouba Google Abderezak Zebboudj Chafia Touil-Boukoffa Mehdi Bourouba Google Scholar Abderezak Zebboudj Chafia Touil-Boukoffa Mehdi Bourouba PubMed Abderezak Zebboudj Chafia Touil-Boukoffa Mehdi Bourouba Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Autophagy of SO-Rb50 cells induced by arsenic trioxide

Background Cellular autophagy is a non-apoptosis death form of tumor tissue. Research determined that arsenic trioxide （As2O3） leads to apoptosis of tumor cells. But whether As2O3 induce autophagy of SO-Rb50 cells or not is unclear. Objective This study was to assess the effects of As2O3 on autophagy of SO-Rh50 cells. Methods As2O3 with the concentration of 0,0. 5,1.0,2.0,4.0 p.mol/L was used to treat the SO-Rb50 cell line for 48 hours,and the growth and proliferation of SO-Rb50 ceils were detected using MTT assay （A570 ） . pGFP- LC3 ,a marker of autophagy,was constructed to transfer SO-Rb50 cells, and the cells were then divided into RPMI- 1640 culture group （ untreated group） , As2O3 ＋ RPMI-1640 culture group （ As2O3 treated group） and rapamycin culture group （positive control group）. Autophagy of SO-Rh50 cells was examined by laser confocal microscope and monodansylcadaverine （MDC） influorescence staining, respectively,48 hours following cell culture, ghrastructural features of autophagy were examined with transmission electron microscope （TEM）. The percentage of autophagy positive cells in different concentrations of As203 treated groups was calculated with flow eytometer. Results The A570 values of SO-Rb50 cells were 2. 194-＋0. 066,1. 841-＋0. 213,1. 035-＋0. 046,0. 374-＋0. 042 and 0. 167-＋0. 019 in 0,0.5,1.0,2.0,4.0 p.mol/L As2 03 treated groups, with a significant difference among these 5 groups （ F = 547. 636,P〈0. 05 ）, and those of 0. 5,1.0,2. 0,4.0 μmol/L As203 treated groups were significantly reduced in comparison with untreated group （P = 0. 000）. The positive granular spots for GFP-LC3 chimeric protein were seen to aggregate in autophagic vacuoles in the As2O3 treated group and positive control group,but diffuse cytoplasmic signal for GFP-LC3 was found in the untreated group. Normal ultrastructure of SO-Rb50 cells was exhibited in the untreated group, and many double-membrane-like bound vesicles and autlysosomes were documented in the As203 treated group and positive control group under the TEM. A lots of MDC fluorescence granule were found in the As2O3 treated group and positive control group rather than the untreated group. Flow cytometry showed that the percentages of SO-Rb50 cells were 0,15.6% ,42.7% ,57.9% ,79.5% and 89.0% in the 0,0.5,1.0,2.0,4.0 μmol/L As2O3 groups and positive control group,respectively,showing a As203 concentration-dependent increase. Conclusions As2O3 can induce the autophagy of SO-Rb50 cells and inhibit the proliferation of SO-Rb50 cells. Autophagic response of SO-Rb50 cells appears prior to the nuclear change after exposed toAs2O3. The degree of autophagy of SO-Rb50 cells is associated with As2O3 dose. Key words: Retinal blastoma; Autophagy; Chemotherapy; Microtubules related protein light chain 3

Role of S100A8/A9 in the cross-talk between cancer cells and stroma-associated cells

s / Pancreatology 12 (2012) 502–597 589 1 Tampere Pancreas Laboratory, Tampere University Hospital, Tampere, Finland Department of Gastroenterology and Alimentary Tract Surgery, Tampere University Hospital, Tampere, Finland Department of Gastroenterological Surgery, Kumamoto University Graduate School of Medical Sciences, Kumamoto, Japan Objectives: Studies with PANC-1 imply that autophagymay be induced in pancreatic ductal adenocarcinoma (PDA). Investigating the role of autophagy in patients is challenging as the induction of autophagy in pancreatic tissue may be affected by cell death mechanisms taking place after the tissue specimens have been harvested. Eliminating this postharvesting distraction would be crucial when investigating this phenomenon. No such study about autophagy in PDA in patients exists. Our aim was to study, whether autophagy is 1) induced in PDA compared to healthy pancreatic tissue in patients, and 2) correspondent to the autophagic status of PANC-1 cells in vitro. Patients and methods: Pancreatic samples were retrieved in laparotomy from patients undergoing pancreato-duodenectomy. Core-needle biopsies from the center of histologically-confirmed PDA tumors and from healthy pancreas (within the area to be resected) were immediately snapfrozen and proteins were extracted by repeated freeze-thaw cycles. The degree of autophagy was analysed by Western blotting of LC3-II, and compared to the degree of autophagy in PANC-1 cells (in basal conditions and when treated with anticancer drugs (gemcitabine and 5-FU) +/autophagy inhibitor chloroquine). Results: Autophagy was significantly induced in PDA compared to healthy pancreatic tissue in patients. This resembled the autophagic status of PANC-1 cells under basal culture conditions. Conclusions: Autophagy is induced in PDA in patients similarly to PANC-1 cells. In PANC-1 cells chloroquine increases the cytotoxicity of 5FU and gemcitabine by inhibiting autophagy. Thus patients with PDA may benefit from therapy where anticancer drugs are combined with an autophagy inhibitor.

Abstract 2037: Anti-miRNA-21 confers radiosensitivity through inhibition of PI3K/AKT pathway and enhancing autophagy

Abstract Radiation is a core part of therapy for malignant glioma, which has been given after debulking surgery and prolonged patient's survival. However, the mass of glioma hardly shows response to radiation and frequently become radioresistant after the initial radiation. Among onco-microRNAs, microRNA-21 regulates expression of various molecules involved in anti-apoptosis, invasion, and metastasis in cancer. Yet, there has been known to the relationship between microRNA-21 and radioresistance of glioma. In this study, we investigate the effect of anti-mir-21 on radiosensitivity in glioblastoma and cervical cancer cell lines. Induction of mature microRNA-21 was confirmed by Nothern blotting after irradiation and the transfection of anti-mir-21 resulted in radiosenstivity on colony forming assay. The radiosensitive is thought to be the results of impairment of DNA repair in irradiated cancer cells, which is proved by γ-H2AX staining. In contrast to control group, a significant percentage of anti-mir-21 transfected cells retain DNA foci at 24 hours after irradiation. The suppression of radiation-activated PI3K/Akt pathway is proved on Western blot and thought to be one of the mechanisms for anti-mir-21 induced radiosensitization. Cell cycle analysis shows significant increase of G2/M phase arrest in anti-mir-21 treated cells compared to the control while subG1 population representing apoptotic cells reveals no significant difference between two groups. To investigate further the responsible cell death mechanism other than apoptosis, we measured the degree of autophagy in irradiated cells by both counting acid vesicular organelles using flow cytometry and Western blotting for LC-3 band. The autophagy is significantly increased in anti-mir-21 treated cells after radiation compared to mock treated cells. We suggest that microRNA-21 is a pivotal regulator of mechanisms to evade radiation-induced cell death in cancer cell. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2037.