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

Influence of zirconium and copper sub-layer in cell integrations on femtosecond laser-processed Ti thin films

The creation of novel biocompatible Ti-based thin films with a Zr or Cu sub-layer modified by ultrafast laser processing is studied. To prepare bioactive surfaces, ultrafast laser processing is focused on the formation of laser-induced periodic surface structures (LIPSS) with the production of oxide phases at the surfaces. Two differently designed multilayer thin films Ti/Cu/Ti and Ti/Zr/Ti were deposited on the silicon using the ion sputtering method. The Ti thin film contains Cu or Zr sub-layer (thickness of 10 nm) at the 10 nm below the surface. The composition and surface morphology variations for these systems, deposited and laser-processed under the same experimental conditions, were caused only by different thermo-physical properties of the sub-layer (Cu or Zr). The surface morphology in the form of LIPSS, led to improved cell adhesion and stable cells/thin films interface compared to as-deposited samples. Field-emission scanning electron microscopy and MTT analysis revealed that laser processing of both systems increased cell adhesion, proliferation, and metabolical activity of L929 mouse fibroblast cells compared to non-modified flat surfaces. Overall, the biocompatibility of Zrcontaining thin films is better than Ti/Cu/Ti system. Further, laser processing and formation of LIPSS makes Ti/Zr/Ti thin films excellent candidate for biomedica

Xanthone-rich extract from Gentiana dinarica transformed roots and its active component norswertianin induce autophagy and ROS-dependent differentiation of human glioblastoma cell line

BACKGROUND Glioblastoma multiforme (GMB) is the most malignant of all brain tumors with poor prognosis. Anticancer potential of xanthones, bioactive compounds found in Gentiana dinarica, is well-documented. Transformation of G. dinarica roots with Agrobacterium rhizogenes provides higher xanthones accumulation, which enables better exploitation of these anticancer compounds. HYPOTHESIS/PURPOSE The aim of this study was to investigate antiglioma effect of three different G. dinarica extracts: E1—derived from untransformed roots, E2—derived from roots transformed using A. rhizogenes strain A4M70GUS, and E3—derived from roots transformed using A. rhizogenes strain 15834/PI. Further, mechanisms involved in anticancer potential of the most potent extract were examined in detail, and its active component was determined. METHODS The cell viability was assessed using MTT and crystal violet test. Cell cycle analysis, the expression of differentiation markers, the levels of autophagy, and oxidative stress were analyzed by flow cytometry. Autophagy and related signaling pathways were assessed by immunoblotting. RESULTS E3, in contrast to E1 and E2, strongly reduced growth of U251 human glioblastoma cells, triggered cell cycle arrest in G2/M phase, changed cellular morphology, and increased expression of markers of differentiated astrocytes (glial fibrillary acidic protein) and neurons (β-tubulin). E3 stimulated autophagy, as demonstrated by enhanced intracellular acidification, increased microtubule-associated light chain 3B (LC3-I) conversion to autophagosome associated LC3-II, and decreased level of selective autophagy target p62. Induction of autophagy was associated with Akt-dependent inhibition of main autophagy suppressor mammalian target of rapamycin (mTOR). Both genetic and pharmacological inhibition of autophagy suppressed the expression of differentiation markers, but had no effect on cell cycle arrest in E3-treated cells. E3 stimulated oxidative stress, and antioxidants vitamin E and N-acetyl cysteine inhibited autophagy and differentiation of E3-treated U251 cells. The most prevalent compound of E3, xanthone aglycone norswertianin, also arrested glioblastoma cell proliferation in G2/M phase and induced glioblastoma cell differentiation through induction of autophagy and oxidative stress. CONCLUSION These results indicate that E3 and its main active component norswertianin may serve as a potential candidate for differentiation therapy of glioblastoma

Influence of the addition of different radiopacifiers and bioactive nano-hydroxyapatite on physicochemical and biological properties of calcium silicate based endodontic ceramic

The purpose of this study was to investigate the influence of different radiopacifiers on the physicochemical and biological properties of novel calcium silicate based endodontic ceramic enriched with bioactive nanoparticulated hydroxyapatite - ECHA. Namely, ECHA was used as a basis for mixing with the following radiopacifiers: strontium fluoride (SrF2), zirconium dioxide (ZrO2) and bismuth oxide (Bi2O3). For comparison, Portland cement (PC) and mineral trioxide aggregate (MTA) were used. The following physicochemical characteristics were examined: the radiopacity, setting time, compressive strength, porosity, wettability and pH value. The biocompatibility of the cements was assessed by crystal violet, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) and cell adhesion assays. The highest radiopacity was obtained for the ECHA + Bi2O3 mixture and MTA that were statistically significant in comparison to other materials (p lt 0.05). Both initial and final setting times as well as compressive strengths were statistically lower for experimental cements than for PC and MTA (p lt 0.05). The lowest total porosity was observed in the ECHA + ZrO2 group when compared with the other two experimental cements (p lt 0.05), but not when compared with PC and MTA (p > 0.05). Experimental cements exhibited statistically higher contact angles of glycerol than PC and MTA (p lt 0.05). For blood plasma, a statistical difference was found only between ECHA + Bi2O3 and PC (p lt 0.05). All investigated materials had alkalization ability. Cell viability assays revealed that the extracts of tested cements did not exhibit cytotoxic effect on L929 cells. Scanning electron microscopy had shown a high degree of cell proliferation and adhesion of cells from apical papilla on experimental cements' surfaces. Novel endodontic ceramics with nano-hydroxyapatite addition have satisfactory biological and physicochemical properties when compared to MTA and PC controls. Considerable lower setting time of experimental cements might present a huge advantage of these synthesized materials in clinical practice. SrF2 presents a novel promising radiopacifying agent for dental cements manufacturing

Multi-target potential of newly designed tacrine-derived cholinesterase inhibitors: Synthesis, computational and pharmacological study

Simple and scalable synthetic approach was used for the preparation of thirteen novel tacrine derivatives consisting of tacrine and N-aryl-piperidine-4-carboxamide moiety connected by a five-methylene group linker. An anti-Alzheimer disease (AD) potential of newly designed tacrine derivatives was evaluated against two important AD targets, acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). In vitro pharmacological evaluation showed strong ChE inhibitory activity of all compounds, with IC50 values ranging from 117.5 to 455 nM for AChE and 34 to 324 nM for BuChE. As a representative of the series with the best cytotoxicity / ChE inhibitory activity ratio, expressed as the selectivity index (SI), 2-chlorobenzoyl derivative demonstrated mixed-type inhibition on AChE and BuChE, suggesting binding to both CAS and PAS of the enzymes. It also exhibited antioxidant capacity and neuroprotective potential against amyloid-β (Aβ) toxicity in the culture of neuron-like cells. In-depth computational analysis corroborated well with in vitro ChE inhibition, illuminating that all compounds exhibit significant potential in targeting both enzymes. Molecular dynamics (MD) simulations revealed that 2-chlorobenzoyl derivative, created complexes with AChE and BuChE that demonstrated sufficient stability throughout the observed MD simulation. Computationally predicted ADME properties indicated that these compounds should have good blood–brain barrier (BBB) permeability, an important factor for CNS-targeting drugs. Overall, all tested compounds showed promising pharmacological behavior, highlighting the multi-target potential of 2- chlorobenzoyl derivative which should be further investigated as a new lead in the drug development process

Influence of the addition of different radiopacifiers and bioactive nano-hydroxyapatite on physicochemical and biological properties of calcium silicate based endodontic ceramic

The purpose of this study was to investigate the influence of different radiopacifiers on the physicochemical and biological properties of novel calcium silicate based endodontic ceramic enriched with bioactive nano-particulated hydroxyapatite – ECHA. Namely, ECHA was used as a basis for mixing with the following radiopacifiers: strontium fluoride (SrF2), zirconium dioxide (ZrO2) and bismuth oxide (Bi2O3). For comparison, Portland cement (PC) and mineral trioxide aggregate (MTA) were used. The following physicochemical characteristics were examined: the radiopacity, setting time, compressive strength, porosity, wettability and pH value. The biocompatibility of the cements was assessed by crystal violet, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) and cell adhesion assays. The highest radiopacity was obtained for the ECHA + Bi2O3 mixture and MTA that were statistically significant in comparison to other materials (p 0.05). Experimental cements exhibited statistically higher contact angles of glycerol than PC and MTA (p < 0.05). For blood plasma, a statistical difference was found only between ECHA + Bi2O3 and PC (p < 0.05). All investigated materials had alkalization ability. Cell viability assays revealed that the extracts of tested cements did not exhibit cytotoxic effect on L929 cells. Scanning electron microscopy had shown a high degree of cell proliferation and adhesion of cells from apical papilla on experimental cements’ surfaces. Novel endodontic ceramics with nano-hydroxyapatite addition have satisfactory biological and physicochemical properties when compared to MTA and PC controls. Considerable lower setting time of experimental cements might present a huge advantage of these synthesized materials in clinical practice. SrF2 presents a novel promising radiopacifying agent for dental cements manufacturing

AMP-activated protein kinase inhibits MPP+-induced oxidative stress and apoptotic death of SH-SY5Y cells through sequential stimulation of Akt and autophagy.

We investigated the interplay between the intracellular energy sensor AMP-activated protein kinase (AMPK), prosurvival kinase Akt, oxidative stress, and autophagy in the cytotoxicity of parkinsonian neurotoxin 1-methyl-4-phenyl piridinium (MPP+) towards SH-SY5Y human neuroblastoma cells. MPP+-mediated oxidative stress, mitochondrial depolarization, and apoptotic cell death were associated with rapid (within 2 h) activation of AMPK, its target Raptor, and prosurvival kinase Akt. Antioxidants N-acetylcysteine and butylated hydroxyanisole suppressed MPP+-induced cytotoxicity, AMPK, and Akt activation. A genetic or pharmacological inhibition of AMPK increased MPP+-triggered production of reactive oxygen species and cell death, and diminished Akt phosphorylation, while AMPK activation protected SH-SY5Y cells from MPP+. On the other hand, genetic or pharmacological inactivation of Akt stimulated MPP+-triggered oxidative stress and neurotoxicity, but did not affect AMPK activation. At later time-points (16-24 h), MPP+ inhibited the main autophagy repressor mammalian target of rapamycin, which coincided with the increase in the levels of autophagy marker microtubule-associated protein 1 light-chain 3B. MPP+ also increased the concentration of a selective autophagic target sequestosome-1/p62 and reduced the levels of lysosomal-associated membrane protein 1 and cytoplasmic acidification, suggesting that MPP+-induced autophagy was coupled with a decrease in autophagic flux. Nevertheless, further pharmacological inhibition of autophagy sensitized SH-SY5Y cells to MPP+-induced death. Antioxidants and AMPK knockdown reduced, whereas genetic inactivation of Akt potentiated neurotoxin-triggered autophagy. These results suggest that MPP+-induced oxidative stress stimulates AMPK, which protects SH-SY5Y cells through early activation of antioxidative Akt and late induction of cytoprotective autophagy

Cyclohexyl Analogues of Ethylenediamine Dipropanoic Acid Induce Caspase-Independent Mitochondrial Apoptosis in Human Leukemic Cells

We investigated the cytotoxicity of recently synthesized (S,S)-ethyleridiamine-N,N'-di-2-(3-cyclohexyl)propanoic acid esters toward human leukemic cell lines and healthy blood mononuclear cells. Cell viability was assessed by acid phosphatase assay, apoptosis, and differentiation were analyzed by flow cytometry and electron microscopy, while intracellular localization of apoptosis-inducing factor (AIF) was determined by immunoblotting. It was demonstrated that methyl, ethyl, and n-propyl esters were toxic to HL-60, REH, MOLT-4, KG-1, JVM-2, and K-562 leukemic cell lines, while the nonesterified parental compound and n-butyl ester were devoid of cytotoxic action. The ethyl ester exhibited the highest cytotoxic activity (IC50 10.7 mu M-45.4 mu M), which was comparable to that of the prototypical anticancer drug cisplatin. The observed cytotoxic effect in HL-60 cells was associated with an increase in superoxide production and mitochondrial membrane depolarization, leading to apoptotic cell death characterized by phosphatidylserine externalization and DNA fragmentation in the absence of autophagic response. DNA fragmentation preceded caspase activation and followed AIF translocation from mitochondria to nucleus, which was indicative of caspase-independent apoptotic cell death. HL-60 cells treated with subtoxic concentration of the compound displayed morphological signs of granulocytic differentiation (nuclear indentations and presence of cytoplasmic primary granules), as well as an increased expression of differentiation markers CD11b and CD15. The cyclohexyl analogues of ethylenediamine dipropanoic acid were also toxic to peripheral blood mononuclear cells of both healthy controls and leukemic patients, the latter being more sensitive. Our data demonstrate that the toxicity of the investigated cyclohexyl compounds against leukemic cell lines is mediated by caspase-independent apoptosis associated with oxidative stress, mitochondrial dysfunction, and AIF translocation

Cyclohexyl Analogues of Ethylenediamine Dipropanoic Acid Induce Caspase-Independent Mitochondrial Apoptosis in Human Leukemic Cells

We investigated the cytotoxicity of recently synthesized (S,S)-ethyleridiamine-N,N'-di-2-(3-cyclohexyl)propanoic acid esters toward human leukemic cell lines and healthy blood mononuclear cells. Cell viability was assessed by acid phosphatase assay, apoptosis, and differentiation were analyzed by flow cytometry and electron microscopy, while intracellular localization of apoptosis-inducing factor (AIF) was determined by immunoblotting. It was demonstrated that methyl, ethyl, and n-propyl esters were toxic to HL-60, REH, MOLT-4, KG-1, JVM-2, and K-562 leukemic cell lines, while the nonesterified parental compound and n-butyl ester were devoid of cytotoxic action. The ethyl ester exhibited the highest cytotoxic activity (IC50 10.7 mu M-45.4 mu M), which was comparable to that of the prototypical anticancer drug cisplatin. The observed cytotoxic effect in HL-60 cells was associated with an increase in superoxide production and mitochondrial membrane depolarization, leading to apoptotic cell death characterized by phosphatidylserine externalization and DNA fragmentation in the absence of autophagic response. DNA fragmentation preceded caspase activation and followed AIF translocation from mitochondria to nucleus, which was indicative of caspase-independent apoptotic cell death. HL-60 cells treated with subtoxic concentration of the compound displayed morphological signs of granulocytic differentiation (nuclear indentations and presence of cytoplasmic primary granules), as well as an increased expression of differentiation markers CD11b and CD15. The cyclohexyl analogues of ethylenediamine dipropanoic acid were also toxic to peripheral blood mononuclear cells of both healthy controls and leukemic patients, the latter being more sensitive. Our data demonstrate that the toxicity of the investigated cyclohexyl compounds against leukemic cell lines is mediated by caspase-independent apoptosis associated with oxidative stress, mitochondrial dysfunction, and AIF translocation.Ministry of Science of the Republic of Serbia [41025, 172035

Cyclohexyl Analogues of Ethylenediamine Dipropanoic Acid Induce Caspase-Independent Mitochondrial Apoptosis in Human Leukemic Cells

We investigated the cytotoxicity of recently synthesized (S,S)-ethyleridiamine-N,N'-di-2-(3-cyclohexyl)propanoic acid esters toward human leukemic cell lines and healthy blood mononuclear cells. Cell viability was assessed by acid phosphatase assay, apoptosis, and differentiation were analyzed by flow cytometry and electron microscopy, while intracellular localization of apoptosis-inducing factor (AIF) was determined by immunoblotting. It was demonstrated that methyl, ethyl, and n-propyl esters were toxic to HL-60, REH, MOLT-4, KG-1, JVM-2, and K-562 leukemic cell lines, while the nonesterified parental compound and n-butyl ester were devoid of cytotoxic action. The ethyl ester exhibited the highest cytotoxic activity (IC50 10.7 mu M-45.4 mu M), which was comparable to that of the prototypical anticancer drug cisplatin. The observed cytotoxic effect in HL-60 cells was associated with an increase in superoxide production and mitochondrial membrane depolarization, leading to apoptotic cell death characterized by phosphatidylserine externalization and DNA fragmentation in the absence of autophagic response. DNA fragmentation preceded caspase activation and followed AIF translocation from mitochondria to nucleus, which was indicative of caspase-independent apoptotic cell death. HL-60 cells treated with subtoxic concentration of the compound displayed morphological signs of granulocytic differentiation (nuclear indentations and presence of cytoplasmic primary granules), as well as an increased expression of differentiation markers CD11b and CD15. The cyclohexyl analogues of ethylenediamine dipropanoic acid were also toxic to peripheral blood mononuclear cells of both healthy controls and leukemic patients, the latter being more sensitive. Our data demonstrate that the toxicity of the investigated cyclohexyl compounds against leukemic cell lines is mediated by caspase-independent apoptosis associated with oxidative stress, mitochondrial dysfunction, and AIF translocation.Ministry of Science of the Republic of Serbia [41025, 172035