T-2 Toxin Induces Apoptotic Cell Death and Protective Autophagy in Mouse Microglia BV2 Cells.
Ontology highlight
ABSTRACT:
SUBMITTER: Sun T
PROVIDER: S-EPMC9330824 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Autophagy is activated by various stresses, including DNA damage, and previous studies of DNA damage-induced autophagy have focused on the response to chemotherapeutic drugs, ionizing radiation, and reactive oxygen species. In this study, we investigated the biological significance of autophagic response to ultraviolet (UV) irradiation in A549 and H1299 cells. Our results indicated that UV induces on-rate autophagic flux in these cells. Autophagy inhibition resulting from the knockdown of beclin-1 and Atg5 reduced cell viability and enhanced apoptosis. Moreover, we found that ATR phosphorylation was accompanied by microtubule-associated protein 1 light chain 3B II (LC3B-II) expression during the early phases following UV irradiation, which is a well-established inducer of ATR. Knocking down ATR further attenuated the reduction in LC3B-II at early stages in response to UV treatment. Despite the potential role of ATR in autophagic response, reduced ATR expression does not affect autophagy induction during late phases (24 and 48 h after UV treatment). The result is consistent with the reduced ATR phosphorylation at the same time points and suggests that autophagic response at this stage is activated via a distinct pathway. In conclusion, this study demonstrated that autophagy acts as a cytoprotective mechanism against UV-induced apoptosis and that autophagy induction accompanied with apoptosis at late stages is independent of ATR activation.
| S-EPMC3269747 | biostudies-literature
Project description:IntroductionMesenchymal stem cells (MSCs) are immunosuppressive, but we lack an understanding of how these adult stem cells are in turn affected by immune cells and the surrounding tissue environment. As MSCs have stromal functions and exhibit great plasticity, the influence of an inflamed microenvironment on their responses is important to determine. MSCs downregulate microglial inflammatory responses, and here we describe the mutual effects of coculturing mouse bone marrow MSCs with BV2 microglia in a lipopolysaccharide (LPS) inflammatory paradigm.MethodsMouse MSCs were cultured from femoral and tibial bone marrow aspirates and characterized. MSCs were cocultured with BV2 microglia at four seeding-density ratios (1:0.2, 1:0.1, 1:0.02, and 1:0.01 (BV2/MSC)), and stimulated with 1 μg/ml LPS. In certain assays, MSCs were separated from BV2 cells with a cell-culture insert to determine the influence of soluble factors on downstream responses. Inflammatory mediators including nitric oxide (NO), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and chemokine (C-C motif) ligand 2 (CCL2) were measured in cocultures, and MSC and BV2 chemotactic ability determined by migration assays.ResultsWe demonstrated MSCs to increase expression of NO and IL-6 and decrease TNF-α in LPS-treated cocultures. These effects are differentially mediated by soluble factors and cell-to-cell contact. In response to an LPS stimulus, MSCs display distinct behaviors, including expressing IL-6 and very high levels of the chemokine CCL2. Microglia increase their migration almost fourfold in the presence of LPS, and interestingly, MSCs provide an equal impetus for microglia locomotion. MSCs do not migrate toward LPS but migrate toward microglia, with their chemotaxis increasing when microglia are activated. Similarly, MSCs do not produce NO when exposed to LPS, but secrete large amounts when exposed to soluble factors from activated microglia. This demonstrates that certain phenotypic changes of MSCs are governed by inflammatory microglia, and not by the inflammatory stimulus. Nonetheless, LPS appears to "prime" the NO-secretory effects of MSCs, as prior treatment with LPS triggers a bigger NO response from MSCs after exposure to microglial soluble factors.ConclusionsThese effects demonstrate the multifaceted and reciprocal interactions of MSCs and microglia within an inflammatory milieu.
| S-EPMC3706938 | biostudies-literature
Project description:BACKGROUND:Microglia play key roles in neuron-glia interaction, neuroinflammation, neural repair, and neurotoxicity. Currently, various microglial in vitro models including primary microglia derived from distinct isolation methods and immortalized microglial cell lines are extensively used. However, the diversity of these existing models raises difficulty in parallel comparison across studies since microglia are sensitive to environmental changes, and thus, different models are likely to show widely varied responses to the same stimuli. To better understand the involvement of microglia in pathophysiological situations, it is critical to establish a reliable microglial model system. METHODS:With postnatal mouse brains, we isolated microglia using three general methods including shaking, mild trypsinization, and CD11b magnetic-associated cell sorting (MACS) and applied RNA sequencing to compare transcriptomes of the isolated cells. Additionally, we generated a genome-wide dataset by RNA sequencing of immortalized BV2 microglial cell line to compare with primary microglia. Furthermore, based on the outcomes of transcriptional analysis, we compared cellular functions between primary microglia and BV2 cells including immune responses to LPS by quantitative RT-PCR and Luminex Multiplex Assay, TGF? signaling probed by Western blot, and direct migration by chemotaxis assay. RESULTS:We found that although the yield and purity of microglia were comparable among the three isolation methods, mild trypsinization drove microglia in a relatively active state, evidenced by high amount of amoeboid microglia, enhanced expression of microglial activation genes, and suppression of microglial quiescent genes. In contrast, CD11b MACS was the most reliable and consistent method, and microglia isolated by this method maintained a relatively resting state. Transcriptional and functional analyses revealed that as compared to primary microglia, BV2 cells remain most of the immune functions such as responses to LPS but showed limited TGF? signaling and chemotaxis upon chemoattractant C5a. CONCLUSIONS:Collectively, we determined the optimal isolation methods for quiescent microglia and characterized the limitations of BV2 cells as an alternative of primary microglia. Considering transcriptional and functional differences, caution should be taken when extrapolating data from various microglial models. In addition, our RNA sequencing database serves as a valuable resource to provide novel insights for appropriate application of microglia as in vitro models.
| S-EPMC5964710 | biostudies-literature
Project description:To survive starvation and other forms of stress, eukaryotic cells undergo a lysosomal process of cytoplasmic degradation known as autophagy. Autophagy has been implicated in a number of cellular and developmental processes, including cell-growth control and programmed cell death. However, direct evidence of a causal role for autophagy in these processes is lacking, resulting in part from the pleiotropic effects of signaling molecules such as TOR that regulate autophagy. Here, we circumvent this difficulty by directly manipulating autophagy rates in Drosophila through the autophagy-specific protein kinase Atg1.We find that overexpression of Atg1 is sufficient to induce high levels of autophagy, the first such demonstration among wild-type Atg proteins. In contrast to findings in yeast, induction of autophagy by Atg1 is dependent on its kinase activity. We find that cells with high levels of Atg1-induced autophagy are rapidly eliminated, demonstrating that autophagy is capable of inducing cell death. However, this cell death is caspase dependent and displays DNA fragmentation, suggesting that autophagy represents an alternative induction of apoptosis, rather than a distinct form of cell death. In addition, we demonstrate that Atg1-induced autophagy strongly inhibits cell growth and that Atg1 mutant cells have a relative growth advantage under conditions of reduced TOR signaling. Finally, we show that Atg1 expression results in negative feedback on the activity of TOR itself.Our results reveal a central role for Atg1 in mounting a coordinated autophagic response and demonstrate that autophagy has the capacity to induce cell death. Furthermore, this work identifies autophagy as a critical mechanism by which inhibition of TOR signaling leads to reduced cell growth.
| S-EPMC1865528 | biostudies-literature
Project description:Butein is a chalcone, a flavonoid that is widely biosynthesized in plants. Butein has been identified to possess varied pharmacological activity and is extractable from traditional Chinese medicinal herbs, therefore applicable for disease treatment. Recently, in vitro and in vivo studies have shown that butein may induce apoptotic cell death in various human cancer cells. In this study we investigated the apoptotic effect of butein and the underlying mechanisms in human cervical cancer cells. Two cell lines, C-33A and SiHa cells, were treated with butein at different dosages for different durations. The effect of butein on cell viability was assessed by MTT assay, which revealed that butein exerted cytotoxicity in both cervical cancer cells in a dose- and time-dependent fashion. Apoptotic pathway-related factors in the butein-treated cervical cancer cells were then examined. JC-1 flow cytometry, cytochrome c assay, and caspase activity assays demonstrated that butein disturbed mitochondrial transmembrane potential, and increased cytosolic cytochrome c levels and caspase activities in both cervical cancer cells. Western blot analysis revealed that butein downregulated anti-apoptotic protein Bcl-xL and led to proteolytic cleavage of poly (ADP-ribose) polymerase. In addition, butein decreased expressions of the inhibitor of apoptosis (IAP) proteins, including X-linked IAP, survivin, and cellular IAP-1. The findings of this study suggest that butein can decrease cervical cancer cell viability via a pro-apoptotic effect, which involves inhibition of the IAP proteins and activation of both extrinsic and intrinsic pro-apoptotic pathways. Therefore, butein may be applicable for cervical cancer treatment.
| S-EPMC6176361 | biostudies-literature
Project description:Autophagy plays important roles in the infection and pathogenesis of many viruses, yet the regulatory roles of autophagy in the process of porcine parvovirus (PPV) infection remain unclear. Herein, we show that PPV infection induces autophagy in porcine placental trophoblasts (PTCs). Induction of autophagy by rapamycin (RAPA) inhibited the occurrence of apoptotic cell death, yet promoted viral replication in PPV-infected cells; inhibition of autophagy by 3-MA or ATG5 knockdown increased cellular apoptosis and reduced PPV replication. Interestingly, we found that in the presence of caspase-inhibitor zVAD-fmk, PPV induces non-apoptotic cell death that was characterized by lysosomal damage and associated with autophagy. Induction of complete autophagy flux by RAPA markedly promoted PPV replication compared with incomplete autophagy induced by RAPA plus bafilomycin (RAPA/BAF) in the early phase of PPV infection (24 h.p.i.). Meanwhile, induction of complete autophagy with RAPA increased lysosomal damage and non-apoptotic cell death in the later phase of PPV infection. Therefore, our data suggest that autophagy can enhance PPV replication and promote the occurrence of lysosomal-damage-associated non-apoptotic cell death in PPV-infected porcine placental trophoblasts.
| S-EPMC7020067 | biostudies-literature
Project description:Tritrichomonas foetus is a serious veterinary pathogen, causing bovine trichomoniasis, a sexually transmitted disease leading to infertility and abortion. T. foetus infects the mucosal surfaces of the reproductive tract. Infection with T. foetus leads to apoptotic cell death of bovine vaginal epithelial cells (BVECs) in culture. An affinity-purified cysteine protease (CP) fraction yielding on sodium dodecyl sulfate-polyacrylamide gel electrophoresis a single band with an apparent molecular mass of 30 kDa (CP30) also induces BVEC apoptosis. Treatment of CP30 with the protease inhibitors TLCK (Nalpha-p-tosyl-l-lysine chloromethyl ketone) and E-64 [l-trans-epoxysuccinyl-leucylamide-(4-guanido)-butane] greatly reduces induction of BVEC apoptosis. Matrix-assisted laser desorption ionization-time-of-flight MALDI-TOF mass spectrometry analysis of CP30 reveals a single peak with a molecular mass of 23.7 kDa. Mass spectral peptide sequence analysis of proteolytically digested CP30 reveals homologies to a previously reported cDNA clone, CP8 (D. J. Mallinson, J. Livingstone, K. M. Appleton, S. J. Lees, G. H. Coombs, and M. J. North, Microbiology 141:3077-3085, 1995). Induction of apoptosis is highly species specific, since the related human parasite Trichomonas vaginalis and associated purified CPs did not induce BVEC death. Fluorescence microscopy along with the Cell Death Detection ELISA(PLUS) assay and flow cytometry analyses were used to detect apoptotic nuclear condensation, DNA fragmentation, and changes in plasma membrane asymmetry in host cells undergoing apoptosis in response to T. foetus infection or incubation with CP30. Additionally, the activation of caspase-3 and inhibition of cell death by caspase inhibitors indicates that caspases are involved in BVEC apoptosis. These results imply that apoptosis is involved in the pathogenesis of T. foetus infection in vivo, which may have important implications for therapeutic interference with host cell death that could alter the course of the pathology in vivo.
| S-EPMC427413 | biostudies-literature
Project description:Serine protease inhibitor Kazal type 3 (Spink3) is a trypsin inhibitor in the pancreas. Spink3-/- pancreatic acinar cells are dead with excessive autophagy at birth (p0.5). To prove the role of nonapoptotic cell death with autophagy, we generated by transgenic technology the pancreas of Spink3-/-;XKI/+ mice contained both normal and dying acinar cells with autophagy. In this new mouse model, chronic inflammation occurred in the pancreas, indicating that some signals from nonapoptotic dead cell induce chronic inflammation in the pancreas. All samples were the pancreas at p0.5. Sample 1 and 2 are the pancreas from wild type (Spink3+/+, control) mice. Sample 3 and 4 are the pancreas from Spink3-/-, which all pancreatic acinar cells show induced nonapoptotic cell death with autophagy. Sample 5 and 6 are the pancreas from Spink3-/-XKI/+, about half acinar cells are normal, but other acinar cells show induced nonapoptotic cell death with autophagy.
2016-07-16 | E-GEOD-48946 | biostudies-arrayexpress
Project description:Successful treatment of oesophageal cancer is hampered by recurrent drug resistant disease. We have previously demonstrated the importance of apoptosis and autophagy for the recovery of oesophageal cancer cells following drug treatment. When apoptosis (with autophagy) is induced, these cells are chemosensitive and will not recover following chemotherapy treatment. In contrast, when cancer cells exhibit only autophagy and limited Type II cell death, they are chemoresistant and recover following drug withdrawal.MicroRNA (miRNA) expression profiling of an oesophageal cancer cell line panel was used to identify miRNAs that were important in the regulation of apoptosis and autophagy. The effects of miRNA overexpression on cell death mechanisms and recovery were assessed in the chemoresistant (autophagy inducing) KYSE450 oesophageal cancer cells.MiR-193b was the most differentially expressed miRNA between the chemosensitive and chemoresistant cell lines with higher expression in chemosensitive apoptosis inducing cell lines. Colony formation assays showed that overexpression of miR-193b significantly impedes the ability of KYSE450 cells to recover following 5-fluorouracil (5-FU) treatment. The critical mRNA targets of miR-193b are unknown but target prediction and siRNA data analysis suggest that it may mediate some of its effects through stathmin 1 regulation. Apoptosis was not involved in the enhanced cytotoxicity. Overexpression of miR-193b in these cells induced autophagic flux and non-apoptotic cell death.These results highlight the importance of miR-193b in determining oesophageal cancer cell viability and demonstrate an enhancement of chemotoxicity that is independent of apoptosis induction.
| S-EPMC4754993 | biostudies-literature
Project description:The present study investigated the role of autophagy, a cellular self-digestion process, in the cytotoxicity of antileukemic drug cytarabine towards human leukemic cell lines (REH, HL-60, MOLT-4) and peripheral blood mononuclear cells from leukemic patients. The induction of autophagy was confirmed by acridine orange staining of intracellular acidic vesicles, electron microscopy visualization of autophagic vacuoles, as well as by the increase in autophagic proteolysis and autophagic flux, demonstrated by immunoblot analysis of p62 downregulation and LC3-I conversion to autophagosome-associated LC3-II in the presence of proteolysis inhibitors, respectively. Moreover, the expression of autophagy-related genes Atg4, Atg5 and Atg7 was stimulated by cytarabine in REH cells. Cytarabine reduced the phosphorylation of the major negative regulator of autophagy, mammalian target of rapamycin (mTOR), and its downstream target p70S6 kinase in REH cells, which was associated with downregulation of mTOR activator Akt and activation of extracellular signal- regulated kinase. Cytarabine had no effect on the activation of mTOR inhibitor AMP-activated protein kinase. Leucine, an mTOR activator, reduced both cytarabine-induced autophagy and cytotoxicity. Accordingly, pharmacological downregulation of autophagy with bafilomycin A1 and chloroquine, or RNA interference-mediated knockdown of LC3? or p62, markedly increased oxidative stress, mitochondrial depolarization, caspase activation and subsequent DNA fragmentation and apoptotic death in cytarabine-treated REH cells. Cytarabine also induced mTOR-dependent cytoprotective autophagy in HL-60 and MOLT-4 leukemic cell lines, as well as primary leukemic cells, but not normal leukocytes. These data suggest that the therapeutic efficiency of cytarabine in leukemic patients could be increased by the inhibition of the mTOR-dependent autophagic response.
| S-EPMC3979773 | biostudies-literature