Extracellular vesicles transfer nuclear Abl-dependent and radiation-induced miR-34c into unirradiated cells to cause bystander effects.

Ionizing radiation (IR) not only activates DNA damage response (DDR) in irradiated cells but also induces bystander effects (BE) in cells not directly targeted by radiation. How DDR pathways activated in irradiated cells stimulate BE is not well understood. We show here that extracellular vesicles secreted by irradiated cells (EV-IR), but not those from unirradiated controls (EV-C), inhibit colony formation in unirradiated cells by inducing reactive oxygen species (ROS). We found that µEV-IR from Abl nuclear localization signal-mutated ( Abl-µNLS) cells could not induce ROS, but expression of wild-type Abl restored that activity. Because nuclear Abl stimulates miR-34c biogenesis, we measured miR-34c in EV and found that its levels correlated with the ROS-inducing activity of EV. We then showed that EV from miR-34c minigene-transfected, but unirradiated cells induced ROS; and transfection with miR-34c-mimic, without radiation or EV addition, also induced ROS. Furthermore, EV-IR from miR34-family triple-knockout cells could not induce ROS, whereas EV-IR from wild-type cells could cause miR-34c increase and ROS induction in the miR-34 triple-knockout cells. These results establish a novel role for extracellular vesicles in transferring nuclear Abl-dependent and radiation-induced miR-34c into unirradiated cells to cause bystander oxidative stress

Modulation of 5-Aminolevulinic acid mediated photodynamic therapy induced cell death in a human lung adenocarcinoma cell line

Photodynamic therapy (PDT) is a cancer treatment involving the administration of a photosensitising drug which selectively accumulates in tumor tissue, followed by irradiation with appropriate wavelength light. It triggers photochemical reactions inducing reactive oxygen species (ROS) production with the consequent cellular damage, which ultimately leads to cell death. Porphyrins are the only photosensitizers (PSs) endogenously synthesized by means of administration of the biological precursor, 5- aminolevulinic acid (ALA). Several antioxidants and ROS scavenger agents: reduced glutathione (GSH), mannitol (Man), l-tryptophan (Trp), ascorbate (Asc) and trolox (Trx), were assayed to determine their ability to modulate ALA-based PDT (ALA-PDT); it was performed on A549 human lung adenocarcinoma cells, by incubating with 1mM ALA for 3 hr and followed by irradiation with or without 1 hr pre-incubation with the modulators. They were previously tested for possible cytotoxicity/ photoactivity in concentrations ranging from 0.01 to 20 mM. The ratio between cell survival after ALA-PDT in the presence and in the absence of the scavenger agent (protection grade: PG) was determined, and the concentration showing no cytotoxicity/ photoactivity and providing the highest PG was used in the subsequent experiments. ALA-PDT alone induced a high percentage of apoptotic cell death (98.4 ± 3.5%) as revealed by acridine orange/ethidium bromide staining and AnnexinV-FITC/propidium iodide labelling. Pre-incubation with the modulators at their highest PG concentration significantly reduced apoptotic cells to 48.3 ± 2.7% (Asc), 58.8 ± 4.2 (Trx), 78.5 ± 3.1% (GSH), 64.3 ± 1.6% (Man), 74.6 ± 2.3% (Trp). ROS involvement in early cell death induction after ALA-PDT was tested by flow cytometry using the fluorescent probes dihydro-dichlorofluorescein diacetate (H2-DCFDA) and methoxyvinylpyrene (MVP) for detection of peroxides and singlet oxygen, respectively. ROS production increased after ALA-PDT (H2-DCFDA positive cells, control: 1.1 ± 0.1 %; 10 min-PDT: 69.3 ± 5.6%; MVP positive cells, control: 0.65 ± 0.35%; 10 min-PDT: 83.5 ± 1.9%). Asc prevented peroxide formation (H2-DCFDA positive cells: 50.7 ± 2.8%) and mostly prevented singlet oxygen increase (MVP positive cells: 25.4 ± 5.2%) whereas Trx limited peroxides formation (H2-DCFDA positive cells: 20.8 ± 0.5%), but did not significantly affected singlet oxygen production (MVP positive cells: 73.6 ± 3.4%). Selective scavenger mediated protection against PDT-induced cell death, and direct detection of specific pro-oxidative agents, entail the strong involvement of ROS in ALA-PDT-mediated tumor eradication, suggesting that undesired photodamage to normal tissue might be attenuated by administration of antioxidant agents.Fil: Teijo, Maria Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Investigaciones sobre Porfirinas y Porfirias. Universidad de Buenos Aires. Centro de Investigaciones sobre Porfirinas y Porfirias; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Diez, Berenice Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Investigaciones sobre Porfirinas y Porfirias. Universidad de Buenos Aires. Centro de Investigaciones sobre Porfirinas y Porfirias; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Battle, A.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Investigaciones sobre Porfirinas y Porfirias. Universidad de Buenos Aires. Centro de Investigaciones sobre Porfirinas y Porfirias; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Fukuda, Haydee. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Investigaciones sobre Porfirinas y Porfirias. Universidad de Buenos Aires. Centro de Investigaciones sobre Porfirinas y Porfirias; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentin

Hyperglycaemia and oxidative stress upregulate HSP60 & HSP70 expression in HeLa cells

Heat Shock Proteins 60 & 70 (HSP60 & HSP70) are intracellular protein that has been shown to be present at elevated levels in systemic circulation in Type 2 Diabetes mellitus (T2DM) patients. Conditions that lead to its secretion, and the mechanism of its translocation from cells, have not yet been defined. The aim of this study was to determine if specific cell stressors associated with T2DM, namely hyperglycaemia and oxidative stress, result in the upregulation of HSP60 in human cells in vitro. Human HeLa cells were grown in media supplemented with 100 mM glucose, 200 μM hydrogen peroxide (H₂O₂), and 50 μM sodium azide. Initially, the effect of these treatments on cell growth rate was examined, with each treatment significantly inhibiting growth rate. LDH and MTT assays were also used to successfully demonstrate that these treatments do not significantly increase cell lysis, but do significantly impair mitochondrial dehydrogenase activity. To confirm this mitochondria specific form of inhibition, DCFDA assay were used to investigate any increases in intracellular reactive oxygen species (ROS) generation. All three treatments resulted in significantly increased ROS generation, with greater ROS production occurring with a greater exposure time. Interestingly, when the protein levels of HSP60 and HSP70 were measured after 3 and 7 days of exposure of the HeLa cells to 100 mM glucose, 200 μM H₂O₂, and 50 μM sodium azide significant induction of these two molecular stress proteins were observed ranging from 2.43-5.08 fold compared to untreated control cells

Mechanisms of modulation of brain microvascular endothelial cells function by thrombin.

Brain microvascular endothelial cells are a critical component of the blood-brain barrier. They form a tight monolayer which is essential for maintaining the brain homeostasis. Blood-derived proteases such as thrombin may enter the brain during pathological conditions like trauma, stroke, and inflammation and further disrupts the permeability of the blood-brain barrier, via incompletely characterized mechanisms. We examined the underlying mechanisms evoked by thrombin in rat brain microvascular endothelial cells (RBMVEC). Our results indicate that thrombin, acting on protease-activated receptor 1 (PAR1) increases cytosolic C

Axitinib induces senescence-associated cell death and necrosis in glioma cell lines: The proteasome inhibitor, bortezomib, potentiates axitinib-induced cytotoxicity in a p21(Waf/Cip1) dependent manner.

Glioblastoma is associated with a poor overall survival despite new treatment advances. Antiangiogenic strategies targeting VEGF based on tyrosine kinase inhibitors (TKIs) are currently undergoing extensive research for the treatment of glioma. Herein we demonstrated that the TKI axitinib induces DNA damage response (DDR) characterized by γ-H2AX phosphorylation and Chk1 kinase activation leading to G2/M cell cycle arrest and mitotic catastrophe in U87, T98 and U251 glioma cell lines. Moreover, we found that p21(Waf1/Cip1) increased levels correlates with induction of ROS and senescence-associated cell death in U87 and T98 cell lines, which are reverted by N-acetyl cysteine pretreatment. Conversely, U251 cell line showed a resistant phenotype in response to axitinib treatment, as evidenced by cell cycle arrest but no sign of cell death. The combinatorial use of axitinib with other therapies, with the aim of inhibiting multiple signaling pathways involved in tumor growth, can increase the efficiency of this TKI. Thus, we addressed the combined effects of axitinib with no toxic doses of the proteasome inhibitor bortezomib on the growth of U87 and T98 axitinib- sensitive and axitinib-resistant U251 cell lines. Compared to single treatments, combined exposure was more effective in inhibiting cell viability of all glioma cell lines, although with different cell death modalities. The regulation of key DDR and cell cycle proteins, including Chk1, γ-H2AX and p21(Waf1/Cip1) was also studied in glioma cell lines. Collectively, these findings provide new perspectives for the use of axitinib in combination with Bortezomib to overcome the therapy resistance in gliomas

Cytotoxic drugs activate KSHV lytic cycle in latently infected PEL cells by inducing a moderate ROS increase controlled by HSF1, NRF2 and p62/SQSTM1

Previous studies have indicated that cytotoxic treatments may induce or not activate viral lytic cycle activation in cancer cells latently infected by Kaposi’s sarcoma-associated herpesvirus (KSHV). To investigate the molecular mechanisms responsible for such an effect, we compared two cytotoxic treatments able to induce the viral lytic cycle, named 12-O-tetradecanoylphorbol 13-acetate (TPA) (T) in combination with sodium butyrate (B) and bortezomib (BZ), with two cytotoxic treatments that did not activate this process, named metformin (MET) and quercetin (Q). Our results indicated that TB and bortezomib increased levels of oxygen reactive species (ROS) while metformin and quercetin reduced them. The finding that N-acetylcysteine (NAC), a reactive oxigen species (ROS) scavenger, counteracted K-bZIP expression induced by TB or bortezomib, confirmed that an ROS increase played a role in KSHV lytic cycle activation. Moreover, we found that TB and bortezomib up-regulated p62/Sequestosome1(p62/SQSTM1) protein, while metformin and quercetin down-regulated it. p62/SQSTM1 silencing or the inhibition of NF-E2-related factor 2 (NRF2) or Heat Shock Factor 1 (HSF1), that mediate p62/SQSTM1 transcription, also reduced KSHV lytic antigen expression induced by TB or bortezomib. Interestingly, such combination treatments further increased intracellular ROS and cytotoxicity induced by the single TB or bortezomib treatment, suggesting that NRF2, HSF1 and p62/SQSTM1 keep the ROS level under control, allowing primary effusion lymphoma (PEL) cells to continue to survive and KSHV to replicate

Cancer epithelia-derived mitochondrial DNA is a targetable initiator of a paracrine signaling loop that confers taxane resistance.

Stromal-epithelial interactions dictate cancer progression and therapeutic response. Prostate cancer (PCa) cells were identified to secrete greater concentration of mitochondrial DNA (mtDNA) compared to noncancer epithelia. Based on the recognized coevolution of cancer-associated fibroblasts (CAF) with tumor progression, we tested the role of cancer-derived mtDNA in a mechanism of paracrine signaling. We found that prostatic CAF expressed DEC205, which was not expressed by normal tissue-associated fibroblasts. DEC205 is a transmembrane protein that bound mtDNA and contributed to pattern recognition by Toll-like receptor 9 (TLR9). Complement C3 was the dominant gene targeted by TLR9-induced NF-κB signaling in CAF. The subsequent maturation complement C3 maturation to anaphylatoxin C3a was dependent on PCa epithelial inhibition of catalase in CAF. In a syngeneic tissue recombination model of PCa and associated fibroblast, the antagonism of the C3a receptor and the fibroblastic knockout of TLR9 similarly resulted in immune suppression with a significant reduction in tumor progression, compared to saline-treated tumors associated with wild-type prostatic fibroblasts. Interestingly, docetaxel, a common therapy for advanced PCa, further promoted mtDNA secretion in cultured epithelia, mice, and PCa patients. The antiapoptotic signaling downstream of anaphylatoxin C3a signaling in tumor cells contributed to docetaxel resistance. The inhibition of C3a receptor sensitized PCa epithelia to docetaxel in a synergistic manner. Tumor models of human PCa epithelia with CAF expanded similarly in mice in the presence or absence of docetaxel. The combination therapy of docetaxel and C3 receptor antagonist disrupted the mtDNA/C3a paracrine loop and restored docetaxel sensitivity

Reactive oxygen species generated in chloroplasts contribute to tobacco leaf infection by the necrotrophic fungus Botrytis cinerea

Reactive oxygen species (ROS) play fundamental roles in plant responses to pathogen infection, including modulation of cell death processes and defense-related gene expression. Cell death triggered as part of the hypersensitive response enhances resistance to biotrophic pathogens, but favors the virulence of necrotrophs. Even though the involvement of ROS in the orchestration of defense responses is well established, the relative contribution of specific subcellular ROS sources to plant resistance against microorganisms with different pathogenesis strategies is not completely known. The aim of this work was to investigate the role of chloroplastic ROS in plant defense against a typical necrotrophic fungus, Botrytis cinerea. For this purpose, we used transgenic Nicotiana tabacum (tobacco) lines expressing a plastid-targeted cyanobacterial flavodoxin (pfld lines), which accumulate lower chloroplastic ROS in response to different stresses. Tissue damage and fungal growth were significantly reduced in infected leaves of pfld plants, as compared with infected wild-type (WT) counterparts. ROS build-up triggered by Botrytis infection and associated with chloroplasts was significantly decreased (70–80%) in pfld leaves relative to the wild type. Phytoalexin accumulation and expression of pathogenesis-related genes were induced to a lower degree in pfld plants than in WT siblings. The impact of fungal infection on photosynthetic activity was also lower in pfld leaves. The results indicate that chloroplast-generated ROS play a major role in lesion development during Botrytis infection. This work demonstrates that the modulation of chloroplastic ROS levels by the expression of a heterologous antioxidant protein can provide a significant degree of protection against a canonical necrotrophic fungus.Fil: Rossi, Franco Rubén. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; ArgentinaFil: Krapp, Adriana del Rosario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Bisaro, Fabiana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina. The Queens University of Belfast; IrlandaFil: Maiale, Santiago Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; ArgentinaFil: Pieckenstain, Fernando Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; ArgentinaFil: Carrillo, Nestor Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentin

Differential bioreactivity of neutral, cationic and anionic polystyrene nanoparticles with cells from the human alveolar compartment: robust response of alveolar type 1 epithelial cells

BACKGROUND: Engineered nanoparticles (NP) are being developed for inhaled drug delivery. This route is non-invasive and the major target; alveolar epithelium provides a large surface area for drug administration and absorption, without first pass metabolism. Understanding the interaction between NPs and target cells is crucial for safe and effective NP-based drug delivery. We explored the differential effect of neutral, cationic and anionic polystyrene latex NPs on the target cells of the human alveolus, using primary human alveolar macrophages (MAC) and primary human alveolar type 2 (AT2) epithelial cells and a unique human alveolar epithelial type I-like cell (TT1). We hypothesized that the bioreactivity of the NPs would relate to their surface chemistry, charge and size as well as the functional role of their interacting cells in vivo. METHODS: Amine- (ANP) and carboxyl- surface modified (CNP) and unmodified (UNP) polystyrene NPs, 50 and 100 nm in diameter, were studied. Cells were exposed to 1–100 μg/ml (1.25-125 μg/cm(2); 0 μg/ml control) NP for 4 and 24 h at 37 °C with or without the antioxidant, N-acetyl cysteine (NAC). Cells were assessed for cell viability, reactive oxygen species (ROS), oxidised glutathione (GSSG/GSH ratio), mitochondrial integrity, cell morphology and particle uptake (using electron microscopy and laser scanning confocal microscopy). RESULTS: ANP-induced cell death occurred in all cell types, inducing increased oxidative stress, mitochondrial disruption and release of cytochrome C, indicating apoptotic cell death. UNP and CNP exhibited little cytotoxicity or mitochondrial damage, although they induced ROS in AT2 and MACs. Addition of NAC reduced epithelial cell ROS, but not MAC ROS, for up to 4 h. TT1 and MAC cells internalised all NP formats, whereas only a small fraction of AT2 cells internalized ANP (not UNP or CNP). TT1 cells were the most resistant to the effects of UNP and CNP. CONCLUSION: ANP induced marked oxidative damage and cell death via apoptosis in all cell types, while UNP and CNP exhibited low cytotoxicity via oxidative stress. MAC and TT1 cell models show strong particle-internalization compared to the AT2 cell model, reflecting their cell function in vivo. The 50 nm NPs induced a higher bioreactivity in epithelial cells, whereas the 100 nm NPs show a stronger effect on phagocytic cells. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12989-015-0091-7) contains supplementary material, which is available to authorized users