Apoptosis Induced by Microtubule Disrupting Drugs in Normal Murine Thymocytes In Vitro

Disruption of cytoplasmic and spindle microtubules by colchicine or nocodazole increases mitotic index, but it also enhances apoptosis in isolated mouse thymocytes; the apoptotic index exceeds 20% after 4 hours of incubation with either drug (5% in controls). Apoptosis was confirmed by DNA fragmentation, and was blocked by calcium chelators and inhibitors of protein synthesis. The apoptotic effect of microtubule disrupting drugs (MOD) was directed to interphase thymocytes and was independent on MOD action on mitotic cells. However, cell death of mitotically arrested cells showed ultrastructural changes similar in many aspects to apoptosis

Graphene quantum dots protect SH-SY5Y neuronal cells from SNP-induced apoptotic death

Introduction: We examined the molecular mechanisms of graphene quantum dot (GQD)- mediated protection of SH-SY5Y human neuroblastoma cells from oxidative/nitrosative stress induced by iron-nitrosyl complex sodium nitroprusside (SNP). Methods: GQD was produced by electrochemical oxidation of graphite and characterized by AFM, UVVIS and FTIR spectroscopy. The antioxidant activity of GQD in cell-free conditions was assessed by DPPH, NBT and EPR analysis. The neuroprotective potential of GQD was determined by cell viability assays MTT, CV. Flow cytometry was used to assess markers of apoptosis and GQD scavenging of intracellular ROS/RNS as well. Cellular internalization of GQD was determined using TEM. Results: GQD prevented SNP-induced apoptosis, caspase activation and mitochondrial depolarization in neuroblastoma cells. Although GQD diminished the NO levels in SNP-treated cells, NO scavengers displayed only a slight protection. GQD significantly protected SH-SY5Y cells from neurotoxicity of lightexhausted SNP, incapable of producing NO, implying that protective mechanism is independent of NO-scavenging. GQD reduced SNP-triggered increase in intracellular levels of ROS, particularly •OH, O2•− in cells and cell-free condition. Nonselective antioxidants, •OH scavengers and iron chelators, mimicked GQD cytoprotection, indicating that GQD protect cells by neutralizing •OH generated in the Fenton reaction. Cellular GQD internalization was required for optimal protection since the removal of extracellular GQD by extensive washing partly diminished their protective effect, suggesting that GQD exerted neuroprotective effect intra- and extracellularly. Conclusion: By demonstrating that GQD protect neuroblastoma cells from SNP-induced apoptosis by •OH/NO scavenging, our results suggest that GQD could be valuable candidates for treatment of neurodegenerative diseases associated with oxidative/nitrosative stress

Effects of Vitamin D3 on the NADPH Oxidase and Matrix Metalloproteinase 9 in an Animal Model of Global Cerebral Ischemia.

Decreased blood flow in the brain leads to a rapid increase in reactive oxygen species (ROS). NADPH oxidase (NOX) is an enzyme family that has the physiological function to produce ROS. NOX2 and NOX4 overexpression is associated with aggravated ischemic injury, while NOX2/4-deficient mice had reduced stroke size. Dysregulation of matrix metalloproteinases (MMPs) contributes to tissue damage. The active form of vitamin D3 expresses neuroprotective, immunomodulatory, and anti-inflammatory effects in the CNS. The present study examines the effects of the vitamin D3 pretreatment on the oxidative stress parameters and the expression of NOX subunits, MMP9, microglial marker Iba1, and vitamin D receptor (VDR), in the cortex and hippocampus of Mongolian gerbils subjected to ten minutes of global cerebral ischemia, followed by 24 hours of reperfusion. The ischemia/reperfusion procedure has induced oxidative stress, changes in the expression of NOX2 subunits and MMP9 in the brain, and increased MMP9 activity in the serum of experimental animals. Pretreatment with vitamin D3 was especially effective on NOX2 subunits, MMP9, and the level of malondialdehyde and superoxide anion. These results outline the significance of the NOX and MMP9 investigation in brain ischemia and the importance of adequate vitamin D supplementation in ameliorating the injury caused by I/R

Apoptosis and appearance of multinuclear C6 glioma cells after treatment by microtubule poisons

Microtubules play a crucial role in a large number of cellular functions, including chromosome separation during cell division. Microtubule poisons, drugs that perturb microtubule function, are used for the treatment of a number of malignant tumors, although the precise mechanisms of their cytotoxic effect are still not well understood. In this study, we have investigated the effects of two microtubule poisons, colchicine and paclitaxel, on rat astrocytoma C6 cell line in vitro. Cells were incubated 24-72 hours with, or without poisons, fixed and processed for analysis by light and electron microscopy. Both type of drugs displayed effects on the microtubule network of C6 glioma cells observed by electron microscopy. Furthermore, microtubule poisons triggered apoptotic cell death, although the extent of apoptosis was rather low, while the number of cells arrested in mitosis was significant (33% after 24h treatment with paclitaxel). The most striking effect of paclitaxel was obesreved after 72h, when over 66% of cells displayed multiple nuclei, implying that glioma cells escape mitotic arrest, that results in formation of multinucleated cells. The results showed that C6 glioma cells are largely resistant to induction of apoptosis by microtubule poisons, so further studies are needed to examine the possibilities to overcome resistance

Native cellulose nanofibrills induce immune tolerance in vitro by acting on dendritic cells

Cellulose nanofibrills (CNFs) are attractive biocompatible, natural nanomaterials for wide biomedical applications. However, the immunological mechanisms of CNFs have been poorly investigated. Considering that dendritic cells (DCs) are the key immune regulatory cells in response to nanomaterials, our aim was to investigate the immunological mechanisms of CNFs in a model of DC-mediated immune response. We found that non-toxic concentrations of CNFs impaired the differentiation, and subsequent maturation of human monocyte-derived (mo)-DCs. In a co-culture with CD4+T cells, CNF-treated mo-DCs possessed a weaker allostimulatory and T helper (Th)1 and Th17 polarizing capacity, but a stronger capacity to induce Th2 cells and CD4+CD25hiFoxP3hi regulatory T cells. This correlated with an increased immunoglobulin-like transcript-4 and indolamine dioxygenase-1 expression by CNF-treated mo-DCs, following the partial internalization of CNFs and the accumulation of CD209 and actin bundles at the place of contacts with CNFs. Cumulatively, we showed that CNFs are able to induce an active immune tolerance by inducing tolerogenic DCs, which could be beneficial for the application of CNFs in wound healing and chronic inflammation therapies

Graphene quantum dot antioxidant and proautophagic actions protect SH-SY5Y neuroblastoma cells from oxidative stress-mediated apoptotic death

We investigated the ability of graphene quantum dot (GQD) nanoparticles to protect SH-SY5Y human neuro blastoma cells from oxidative/nitrosative stress induced by iron-nitrosyl complex sodium nitroprusside (SNP). GQD reduced SNP cytotoxicity by preventing mitochondrial depolarization, caspase-2 activation, and subsequent apoptotic death. Although GQD diminished the levels of nitric oxide (NO) in SNP-exposed cells, NO scavengers displayed only a slight protective effect, suggesting that NO quenching was not the main protective mechanism of GQD. GQD also reduced SNP-triggered increase in the intracellular levels of hydroxyl radical ( • OH), superoxide anion (O2 •− ), and lipid peroxidation. Nonselective antioxidants, • OH scavenging, and iron chelators, but not superoxide dismutase, mimicked GQD cytoprotective activity, indicating that GQD protect cells by neutralizing • OH generated in the presence of SNP-released iron. Cellular internalization of GQD was required for optimal protection, since a removal of extracellular GQD by extensive washing only partly diminished their protective effect. Moreover, GQD cooperated with SNP to induce autophagy, as confirmed by the inhibition of autophagy limiting Akt/PRAS40/mTOR signaling and increase in autophagy gene transcription, protein levels of proauto phagic beclin-1 and LC3-II, formation of autophagic vesicles, and degradation of autophagic target p62. The antioxidant activity of GQD was not involved in autophagy induction, as antioxidants N-acetylcysteine and dimethyl sulfoxide failed to stimulate autophagy in SNP-exposed cells. Pharmacological inhibitors of early (wortmannin, 3-methyladenine) or late stages of autophagy (NH4Cl) efficiently reduced the protective effect of GQD. Therefore, the ability of GQD to prevent the in vitro neurotoxicity of SNP depends on both • OH/NO scavenging and induction of cytoprotective autophagy