Amount Of LC3-II Research Articles

The effects of fipronil exposure on oxidative stress, non-specific immunity, autophagy, and apoptosis in the common carp.

The increase in the area treated with the insecticide fipronil has caused concern for aquatic organisms such as fish. Here, we assessed the effect of fipronil on carp indexes of non-specific immunity, oxidative stress, autophagy, and apoptosis following exposure to 0.074 mg/L and 0.185 mg/L of fipronil in the aqueous environment for 1 day, 3 days, 5 days, and 7 days. It was found that glutathione (GSH), malonaldehyde (MDA), and superoxide dismutase (SOD) in gills were significantly reduced (P < 0.05). The increase in exposure time increases the impact on GSH, SOD, and MDA parameters in the liver and intestine. Liver acid phosphatase (ACP), alkaline phosphatase (AKP), and lysozyme (LZM) activity levels increased significantly in the treatment group on the first day after exposure, except for the 0.074 mg/L group of ACP (P < 0.05). The mRNA expression levels of autophagy-related genes ATG12, ATG5, ATG16L, LC3-II, and BECN1 were generally elevated in the liver and intestine during the initial exposure period (P < 0.05), while mTOR was significantly reduced on the first and third days after treatment (P < 0.05). From the results of Western blotting (WB), we can see that the amount of LC3-II was significantly higher than that of LC3-I at 1, 3, and 5 days of exposure (P < 0.05). Furthermore, the apoptosis-related gene Bcl-2 reached its peak in the liver, intestine, and gill on the first day, and caspase3 was significantly downregulated throughout the exposure period (P < 0.05). The results showed that fipronil was potentially harmful to carp and should be used moderately to reduce the damage to aquatic ecosystems. This study complements the mechanism theory of fipronil on fish toxicology and has a certain value for human health risk assessment.

Abstract 2093: Drug induced autophagy and transcriptional gene expression signatures in breast cancer cell lines

Abstract Cancer cells can bypass the desired cell death response to chemotherapy through cytoprotective autophagy. In support of this hypothesis, drug resistant clones display higher basal autophagy than the correspondent drug sensitive cell lines. Autophagy therefore represents a potential target to reduce drug resistance and to enhance drug efficacy. We induced autophagy in several breast cancer cell lines with differing ER/PR/Her2 status by exposing them to tamoxifen (MCF-7/ER positive cells), trastuzumab (SKBR3/Her2 positive cells), bortezomib and rapamycin (MDA-MB-231 triple negative cells) in the presence and absence of an autophagy inhibitor such as hydroxychloroquine. While the ongoing autophagy flux process was confirmed by the amount of LC3II at the lipidated protein level, total RNA was extracted at the same time to perform RNA sequencing analysis. We performed differential expression analysis between all the drug treatments and the relative basal level of each cell type obtaining a cell and drug-specific gene list. We then compared all the gene lists to find a common, cell and drug-independent signature. Bioinformatic analysis was performed using the software IPA (Ingenuity Pathway Analysis) and setting a threshold of 0.01 for the p value. This analysis suggested a set of canonical pathways that are significantly and commonly affected in all the samples tested. The top canonical pathways disrupted in all the gene sets were the one identified as tRNA charging, CHK proteins in cell cycle check point control, hypoxia signaling and unfolded protein response. We plan to further identify the smallest gene list that is associated with the disruption of all these pathways across all the cells. This signature may help to uncover a consistent biomarker of autophagy that can be used during preclinical and clinical drug development aiming to reduce or eliminate drug resistance and improve cancer treatment outcomes. Citation Format: Francesca Mascia, Kostantinos Karagiannis, Wells W. Wu, Rong-Fong Shen, Ashutosh Rao. Drug induced autophagy and transcriptional gene expression signatures in breast cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2093.

Autophagy associated cytotoxicity and cellular uptake mechanisms of bismuth nanoparticles in human kidney cells

Bismuth compounds have been used for treatment of bacterial infection, and recently bismuth nanoparticles (BiNP) were synthesized for imaging and diagnostic purpose, while safety concern of bismuth cannot be ignored. Here, we prepared ultrasmall BiNP and showed an enhanced tumor imaging, but BiNP revealed a differentiated cytotoxicity in human embryonic kidney 293 cells (HEK293) compared to other cell types. For the first time, we found that BiNP can induce autophagy, shown as the increase of monodansylcadaverine fluorescence staining and the amount of LC3II that can be inhibited by 3-MA. BiNP were capable of entering cells in a dose and time dependent manner by fluorescence and element detection methods BiNP were found to be localized in the cytoplasm observed by transmission electron microscopy and intracellular bismuth element confirmed by energy dispersive X-ray analysis. Using endocytic inhibitors, BiNP were found to require ATP and endosomal trafficking pathways for their cellular uptake. Internalized BiNP did not co-localize with EEA1, but co-localized with Lysotracker/LAMP1/LAMP2 at late time points, indicating BiNP may be retained in the non-early endosomal vacuoles and late endosomes. With our novel finding of bismuth induced autophagy and endocytic mechanisms, potential approaches may be applied to reduce the toxicity by bismuth.

622. Oncolytic Adenoviruses Loaded With Active Drugs as a Novel Drug Delivery System for Cancer Therapy

L-carnosine (β-Ala-His) is a naturally occurring histidine dipeptide, normally found in brain, kidney and in large amounts in muscle. L-carnosine has biological functions, including antioxidant activity, ability to chelate metal ions, as well as anti-inflammatory and anti-senescence properties. Recent studies have demonstrated that 50-100 mM of L-carnosine decreases cell proliferation in a colon cancer cell line HCT116, bearing a mutation in codon 13 of the RAS proto-oncogene. In addition, pre-treatment with L-carnosine decreases the intracellular concentration of Adenosine Triphosphate (ATP) and Reactive Oxygen Species (ROS) and inhibits the cell cycle progression in the G1 phase. The proto-oncogene KRAS is mutated in a wide array of human cancers and is important both in tumour progression and resistance to anticancer drugs. To overcome treatment limitations due to the high intracellular concentration required we have hypothesized that L-carnosine can be conjugated on the capsid of oncolytic viruses. Oncolytic viruses are viruses that are able to replicate specifically in and destroy tumor cells and this property is either inherent or genetically-engineered. The association of viruses with specific drugs, would increase the efficacy of the treatment of human neoplasia due to the synergistic action of virus and drug. First we have developed a strategy to conjugate peptides on viral capsid, based on electrostatic interaction. Then, using different cancer cell lines we found that oncolytic virus coated with L-carnosine with a tail of positively charged polylysine was able to enhance a positive anticancer synergistic effect. Finally, in order to investigate the molecular mechanisms underlying the effect of tumor reduction by oncolytic virus coated with modified L-carnosine, we have used three different approaches. First, we have examined, in samples with virus alone, or in combination with L-carnosine, the oncolytic replication by evaluating the E1A expression, second the apoptotic mechanism by expression of specific genes and at end the autophagy regulation via the amount of LC3-II. In conclusion, we have developed a model to use oncolytic adenovirus as a scaffold to deliver active drugs. Once validated the proposed model could be used as a novel drug delivery system for cancer therapy.

Phosphorylation of the exchange factor DENND3 by ULK in response to starvation activates Rab12 and induces autophagy.

Unc-51-like kinases (ULKs) are the most upstream kinases in the initiation of autophagy, yet the molecular mechanisms underlying their function are poorly understood. We report a new role for ULK in the induction of autophagy. ULK-mediated phosphorylation of the guanine nucleotide exchange factor DENND3 at serines 554 and 572 upregulates its GEF activity toward the small GTPase Rab12. Through binding to LC3 and associating with LC3-positive autophagosomes, active Rab12 facilitates autophagosome trafficking, thus establishing a crucial role for the ULK/DENND3/Rab12 axis in starvation-induced autophagy.

Inhibition of HIF-1α Affects Autophagy Mediated Glycosylation in Oral Squamous Cell Carcinoma Cells.

Purpose. To validate the function of autophagy with the regulation of hypoxia inhibitor-induced glycosylation in oral squamous cell carcinoma cell. Methods. Human Tca8113 cell line was used to detect autophagy and glycosylation related protein expression by western blotting and immunofluorescence with HIF-1α inhibitor. Short interfering RNA (siRNA) transfection blocked human ATG12 and ATG1. Results. HIF-1α inhibitor PX-478 reduced the amount of LC3-II and LC3-I in Tca8113 cells. PX-478 decreased the expression of O-GlcNAc and OGT and increased OGA expression. The tendency of O-GlcNAc showed a similar pattern to OGT. PX-478 gradually decreased OGT expression in Tca8113 cells. Protein level of O-GlcNAc and OGT increased in ATG12 and ATG1 depletion. The expression of OGT decreased at first and then rose slowly with the treatment of Atg12 and Atg1 siRNA and PX-478 fluctuant. Autophagy affected the stability of OGT when HIF-1α signaling was blocked. Conclusions. Autophagy reduced by hypoxic stress inhibited. HIF-1α inhibitor decreased glycosylation. OGT became unstable in the absence of autophagy when HIF-1α signaling was blocked.

A Salmonella enterica conjugative plasmid impairs autophagic flux in infected macrophages

pRST98 was originally isolated from Salmonella enterica serovar typhi and could be transferred among enteric bacilli by conjugation. Our previous studies indicated that it could intervene in autophagy of host cells, while the mechanism remained undefined. Here, we explored how pRST98 influenced the autophagic flux of murine macrophage-like cell line (J774A.1). S. enterica serovar typhimurium wild type strain (χ3306), harboring a 100 kb virulence plasmid, was used as a positive control. pRST98 was transferred into χ3306 virulence plasmid cured strain (χ3337) to create the transconjugant strain (χ3337/pRST98). The bacterial strains incubated with J774A.1 revealed that survival rate of intracellular bacteria carrying pRST98 was higher than that of plasmid free strain; presence of pRST98 decreased the number of autophagy vacuoles, LC3 positive and p62 positive bacteria, and also the level of LC3-II and degradation of p62 in macrophages. After intervention with autophagy inhibitor chloroquine, the amount of LC3-II and autophagy vacuoles were still lower in macrophages infected with strains carrying pRST98. Our study suggested that pRST98 could block or delay the formation of autophagosome in the earlier autophagy process, but couldn't affect the function of autolysosome. This finding provided novel insights into the role of enteric conjugation plasmid in bacterial pathogenesis.

Age-related disruption of autophagy in dermal fibroblasts modulates extracellular matrix components

Autophagy is an intracellular degradative system that is believed to be involved in the aging process. The contribution of autophagy to age-related changes in the human skin is unclear. In this study, we examined the relationship between autophagy and skin aging. Transmission electron microscopy and immunofluorescence microscopy analyses of skin tissue and cultured dermal fibroblasts derived from women of different ages revealed an increase in the number of nascent double-membrane autophagosomes with age. Western blot analysis showed that the amount of LC3-II, a form associated with autophagic vacuolar membranes, was significantly increased in aged dermal fibroblasts compared with that in young dermal fibroblasts. Aged dermal fibroblasts were minimally affected by inhibition of autophagic activity. Although lipofuscin autofluorescence was elevated in aged dermal fibroblasts, the expression of Beclin-1 and Atg5—genes essential for autophagosome formation—was similar between young and aged dermal fibroblasts, suggesting that the increase of autophagosomes in aged dermal fibroblasts was due to impaired autophagic flux rather than an increase in autophagosome formation. Treatment of young dermal fibroblasts with lysosomal protease inhibitors, which mimic the condition of aged dermal fibroblasts with reduced autophagic activity, altered the fibroblast content of type I procollagen, hyaluronan and elastin, and caused a breakdown of collagen fibrils. Collectively, these findings suggest that the autophagy pathway is impaired in aged dermal fibroblasts, which leads to deterioration of dermal integrity and skin fragility.

Salvianolic acid B inhibits autophagy and protects starving cardiac myocytes

To investigate the protective or lethal role of autophagy and the effects of Salvianolic acid B (Sal B) on autophagy in starving myocytes. Cardiac myocytes were incubated under starvation conditions (GD) for 0, 1, 2, 3, and 6 h. Autophagic flux in starving cells was measured via chloroquine (3 μmol/L). After myocytes were treated with Sal B (50 μmol/L) in the presence or absence of chloroquine (3 μmol/L) under GD 3 h, the amount of LC3-II, the abundance of LC3-positive fluorescent dots in cells, cell viability and cellular ATP levels were determined using immunoblotting, immunofluorescence microscopy, MTT assay and luminometer, respectively. Moreover, electron microscopy (EM) and immunofluorescent duel labeling of LC3 and Caspase-8 were used to examine the characteristics of autophagy and apoptosis. Immunoblot analysis showed that the amount of LC3-II in starving cells increased in a time-dependent manner accompanied by increased LC3-positive fluorescence and decreased cell viability and ATP content. Sal B (50 μmol/L) inhibited the increase in LC3-II, reduced the abundance of LC3 immunofluorescence and intensity of Caspase-8 fluorescence, and enhanced cellular viability and ATP levels in myocytes under GD 3 h, regardless of whether chloroquine was present. Autophagy induced by starvation for 3 h led to cell injury. Sal B protected starving cells by blocking the early stage of autophagic flux and inhibiting apoptosis that occurred during autophagy.

Kainic acid induces early and transient autophagic stress in mouse hippocampus

Kainic acid (KA) treatment is a well-established model of hippocampal neuron death mediated in large part by KA receptor-induced excitotoxicity. KA-induced, delayed neuron death has been shown previously to follow the induction of seizures and exhibit characteristics of both apoptosis and necrosis. Growing evidence supports a role of autophagic stress-induced death of neurons in several in vitro and in vivo models of neuron death and neurodegeneration. However, whether autophagic stress also plays a role in KA-induced excitotoxicity has not been previously investigated. To examine whether KA alters the levels of proteins associated with or known to regulate the formation of autophagic vacuoles, we isolated hippocampal extracts from control mice and in mice following 2–16h KA injection. KA induced a significant increase in the amount of LC3-II, a specific marker of autophagic vacuoles, at 4–6h following KA, which indicates a transient induction of autophagic stress. Levels of autophagy-associated proteins including ATG5 (conjugated to ATG12), ATG6 and ATG7 did not change significantly after treatment with KA. However, ratios of phospho-mTOR/mTOR were elevated from 6 to 16h, and ratios of phospho-Akt/Akt were elevated at 16h following KA treatment, suggesting a potential negative feedback loop to inhibit further stimulation of autophagic stress. Together these data indicate the transient induction of autophagic stress by KA which may serve to regulate excitotoxic death in mouse hippocampus.

Lysosomal Turnover, but Not a Cellular Level, of Endogenous LC3 is a Marker for Autophagy

During starvation-induced autophagy in mammals, autophagosomes form and fuse with lysosomes, leading to the degradation of the intra-autophagosomal contents by lysosomal proteases. During the formation of autophagosomes, LC3 is lipidated, and this LC3-phospholipid conjugate (LC3-II) is localized on autophagosomes and autolysosomes. While intra-autophagosomal LC3-II may be degraded by lysosomal hydrolases, recent studies have regarded LC3-II accumulation as marker of autophagy. The effect of lysosomal turnover of endogenous LC3-II in this process, however, has not been considered. We therefore investigated the lysosomal turnover of endogenous LC3-II during starvation-induced autophagy using E64d and pepstatin A, which inhibit lysosomal proteases, including cathepsins B, D, and L. We found that endogenous LC3-II significantly accumulated in the presence of E64d and pepstatin A under starvation conditions, increasing about 3.5 fold in HEK293 cells and about 6.7 fold in HeLa cells compared with that in their absence, whereas the amount of LC3-II in their absence is cell-line dependent. Morphological analyses indicated that endogenous LC3-positive puncta and autolysosomes increased in HeLa cells under starvation conditions in the presence of these inhibitors. These results indicate that endogenous LC3-II is considerably degraded by lysosomal hydrolases after formation of autolysosomes, and suggest that lysosomal turnover, not a transient amount, of this protein reflects starvation-induced autophagic activity.