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 LC3β 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.
The discovery of cisplatin and the subsequent research revealed the importance of dinitrogen-containing moiety for the anticancer action of metal complexes. Moreover, certain diamine ligands alone ...display cytotoxicity that contributes to the overall activity of corresponding complexes.
To summarize the current knowledge on the anticancer efficacy, selectivity, and the mechanisms of action of metal complexes with various types of diamine ligands.
The contribution of aliphatic acyclic, aliphatic cyclic, and aromatic diamine ligands to the anticancer activity and selectivity/toxicity of metal complexes with different metal ions were analyzed by comparison with organic ligand alone and/or conventional platinum-based chemotherapeutics.
The aliphatic acyclic diamine ligands are present mostly in complexes with platinum. Aliphatic cyclic diamines are part of Pt(II), Ru(II) and Au(III) complexes, while aromatic diamine ligands are found in Pt(II), Ru(II), Pd(II) and Ir(III) complexes. The type and oxidation state of metal ions greatly influences the cytotoxicity of metal complexes with aliphatic acyclic diamine ligands. Lipophilicity of organic ligands, dependent on alkyl-side chain length and structure, determines their cellular uptake, with edda and eddp/eddip ligands being most useful in this regard. Aliphatic cyclic diamine ligands improved the activity/toxicity ratio of oxaliplatin-type complexes. The complexes with aromatic diamine ligands remain unexplored regarding their anticancer mechanism. The investigated complexes mainly caused apoptotic or necrotic cell death.
Metal complexes with diamine ligands are promising candidates for efficient and more selective alternatives to conventional platinum-based chemotherapeutics. Further research is required to reveal the chemico-physical properties and molecular mechanisms underlying their biological activity.
Metformin is an antidiabetic drug with anticancer properties, which mainly acts through induction of AMP-activated protein kinase (AMPK). In the present study we investigated the influence of ...metformin on the in vitro anticancer activity of the well-known chemotherapeutic agent cisplatin. Cell viability was determined by MTT and LDH release assay, oxidative stress and apoptosis (caspase activation, DNA fragmentation, and phosphatidylserine exposure) were assessed by flow cytometry, while activation of AMPK and Akt was analyzed by immunoblotting. Although metformin reduced the number of tumour cells when applied alone, it surprisingly antagonized the cytotoxicity of cisplatin towards U251 human glioma, C6 rat glioma, SHSY5Y human neuroblastoma, L929 mouse fibrosarcoma and HL-60 human leukemia cell lines. Only in B16 mouse melanoma cells metformin augmented the cytotoxicity of cisplatin. In U251 glioma cells metformin suppressed cisplatin-induced apoptotic cell death through inhibition of oxidative stress and caspase activation. The observed cytoprotection was apparently AMPK-independent, as metformin did not further increase cisplatin-induced AMPK activation in U251 cells and other pharmacological AMPK activators failed to block cisplatin-mediated apoptosis. On the other hand, metformin induced Akt activation in cisplatin-treated cells and Akt inhibitor 10-DEBC hydrochloride or phosphoinositide 3-kinase/Akt inhibitor LY294002 abolished metformin-mediated antioxidant and antiapoptotic effects. In conclusion, the antidiabetic drug metformin reduces cisplatin in vitro anticancer activity through AMPK-independent upregulation of Akt survival pathway. These data warrant caution when considering metformin for treatment of diabetic cancer patients receiving cisplatin or as a potential adjuvant in cisplatin-based chemotherapeutic regimens.
In this review we analyze the ability of antipsychotic medications to modulate macroautophagy, a process of controlled lysosomal digestion of cellular macromolecules and organelles. We focus on its ...molecular mechanisms, consequences for the function/survival of neuronal and other cells, and the contribution to the beneficial and side-effects of antipsychotics in the treatment of schizophrenia, neurodegeneration, and cancer. A wide range of antipsychotics was able to induce neuronal autophagy as a part of the adaptive stress response apparently independent of mammalian target of rapamycin and dopamine receptor blockade. Autophagy induction by antipsychotics could contribute to reducing neuronal dysfunction in schizophrenia, but also to the adverse effects associated with their long-term use, such as brain volume loss and weight gain. In neurodegenerative diseases, antipsychotic-stimulated autophagy might help to increase the clearance and reduce neurotoxicity of aggregated proteotoxins. However, the possibility that some antipsychotics might block autophagic flux and potentially contribute to proteotoxin-mediated neurodegeneration must be considered. Finally, the anticancer effects of autophagy induction by antipsychotics make plausible their repurposing as adjuncts to standard cancer therapy.
We investigated the role of autophagy, a controlled lysosomal degradation of cellular macromolecules and organelles, in glutamate excitotoxicity during nutrient deprivation in vitro. The incubation ...in low-glucose serum/amino acid-free cell culture medium synergized with glutamate in increasing AMP/ATP ratio and causing excitotoxic necrosis in SH-SY5Y human neuroblastoma cells. Glutamate suppressed starvation-triggered autophagy, as confirmed by diminished intracellular acidification, lower LC3 punctuation and LC3-I conversion to autophagosome-associated LC3-II, reduced expression of proautophagic beclin-1 and ATG5, increase of the selective autophagic target NBR1, and decreased number of autophagic vesicles. Similar results were observed in PC12 rat pheochromocytoma cells. Both glutamate-mediated excitotoxicity and autophagy inhibition in starved SH-SY5Y cells were reverted by NMDA antagonist memantine and mimicked by NMDA agonists D-aspartate and ibotenate. Glutamate reduced starvation-triggered phosphorylation of the energy sensor AMP-activated protein kinase (AMPK) without affecting the activity of mammalian target of rapamycin complex 1, a major negative regulator of autophagy. This was associated with reduced mRNA levels of autophagy transcriptional activators (FOXO3, ATF4) and molecules involved in autophagy initiation (ULK1, ATG13, FIP200), autophagosome nucleation/elongation (ATG14, beclin-1, ATG5), and autophagic cargo delivery to autophagosomes (SQSTM1). Glutamate-mediated transcriptional repression of autophagy was alleviated by overexpression of constitutively active AMPK. Genetic or pharmacological AMPK activation by AMPK overexpression or metformin, as well as genetic or pharmacological autophagy induction by TFEB overexpression or lithium chloride, reduced the sensitivity of nutrient-deprived SH-SY5Y cells to glutamate excitotoxicity. These data indicate that transcriptional inhibition of AMPK-dependent cytoprotective autophagy is involved in glutamate-mediated excitotoxicity during nutrient deprivation in vitro.
The aim of our study was to employ fractal analysis for evaluation of ultrastructural changes during early stages of apoptosis. Apoptosis was induced in U251 human glioma cell line by exposure to UVB ...light. The cells were visualized by optical phase-contrast microscopy and photographed before the UV treatment, immediately after the treatment, as well as at 30min intervals during 5h observation period. For each of the 32 cells analyzed, cellular and nuclear fractal dimension, as well as nuclear lacunarity, were determined at each time point. Our data demonstrate that cellular ultrastructural complexity determined by fractal dimension and lacunarity significantly decreases after the UV irradiation, with the nuclear lacunarity being a particularly sensitive parameter in detecting early apoptosis. Importantly, fractal analysis was able to detect cellular apoptotic changes earlier than conventional flow cytometric analysis of phosphatidylserine exposure, DNA fragmentation and cell membrane permeabilization. These results indicate that fractal analysis might be a powerful and affordable method for non-invasive early identification of apoptosis in cell cultures.
► We use fractal analysis to evaluate ultrastructural changes in UV-induced apoptosis. ► Cell images obtained by optical microscopy were subjected to fractal analysis. ► Celullar fractal dimension and nuclear lacunarity decrease in apoptotic cells. ► Fractal analysis detects apoptotic changes earlier than the conventional methods. ► Fractal analysis is an affordable method for early detection of apoptosis.
The mechanisms underlying the cytotoxic action of pure fullerene suspension (nano-C60) and water-soluble polyhydroxylated fullerene C60(OH)n were investigated. Crystal violet assay for cell viability ...demonstrated that nano-C60 was at least three orders of magnitude more toxic than C60(OH)n to mouse L929 fibrosarcoma, rat C6 glioma, and U251 human glioma cell lines. Flow cytometry analysis of cells stained with propidium iodide (PI), PI/annexin V–fluorescein isothiocyanate, or the redox-sensitive dye dihydrorhodamine revealed that nano-C60 caused rapid (observable after few hours), reactive oxygen species (ROS)–associated necrosis characterized by cell membrane damage without DNA fragmentation. In contrast, C60(OH)n caused delayed, ROS-independent cell death with characteristics of apoptosis, including DNA fragmentation and loss of cell membrane asymmetry in the absence of increased permeability. Accordingly, the antioxidant N-acetylcysteine protected the cell lines from nano-C60 toxicity, but not C60(OH)n toxicity, while the pan-caspase inhibitor z-VAD-fmk blocked C60(OH)n-induced apoptosis, but not nano-C60–mediated necrosis. Finally, C60(OH)n antagonized, while nano-C60 synergized with, the cytotoxic action of oxidative stress–inducing agents hydrogen peroxide and peroxynitrite donor 3-morpholinosydnonimine. Therefore, unlike polyhydroxylated C60 that exerts mainly antioxidant/cytoprotective and only mild ROS-independent pro-apoptotic activity, pure crystalline C60 seems to be endowed with strong pro-oxidant capacity responsible for the rapid necrotic cell death.
As autophagy can promote or inhibit inflammation, we examined autophagy-inflammation interplay in COVID-19. Autophagy markers in the blood of 19 control subjects and 26 COVID-19 patients at hospital ...admission and one week later were measured by ELISA, while cytokine levels were examined by flow cytometric bead immunoassay. The antiviral IFN-α and proinflammatory TNF, IL-6, IL-8, IL-17, IL-33, and IFN-γ were elevated in COVID-19 patients at both time points, while IL-10 and IL-1β were increased at admission and one week later, respectively. Autophagy markers LC3 and ATG5 were unaltered in COVID-19. In contrast, the concentration of autophagic cargo receptor p62 was significantly lower and positively correlated with TNF, IL-10, IL-17, and IL-33 at hospital admission, returning to normal levels after one week. The expression of SARS-CoV-2 proteins NSP5 or ORF3a in THP-1 monocytes caused an autophagy-independent decrease or autophagy-inhibition-dependent increase, respectively, of intracellular/secreted p62, as confirmed by immunoblot/ELISA. This was associated with an NSP5-mediated decrease in TNF/IL-10 mRNA and an ORF3a-mediated increase in TNF/IL-1β/IL-6/IL-10/IL-33 mRNA levels. A genetic knockdown of p62 mimicked the immunosuppressive effect of NSP5, and a p62 increase in autophagy-deficient cells mirrored the immunostimulatory action of ORF3a. In conclusion, the proinflammatory autophagy receptor p62 is reduced inacute COVID-19, and the balance between autophagy-independent decrease and autophagy blockade-dependent increase of p62 levels could affect SARS-CoV-induced inflammation.
The role of autophagy, a process in which the cell self-digests its own components, was investigated in glioma cell death induced by the hydroxymethylglutaryl-coenzyme A (HMG-CoA) ...reductase-inhibiting drug simvastatin. Induction of autophagy and activation of autophagy-regulating signalling pathways were analyzed by immunoblotting. Flow cytometry/fluorescent microscopy was used to assess autophagy-associated intracellular acidification and apoptotic markers (phosphatidylserine exposure, DNA fragmentation and caspase activation). Cell viability was determined by crystal violet, MTT or LDH release assay. Simvastatin treatment of U251 and C6 glioma cell lines caused the appearance of autophagolysosome-like intracytoplasmic acidic vesicles. The induction of autophagy in U251 cells was confirmed by the upregulation of autophagosome-associated LC3-II and pro-autophagic beclin-1, as well as by the downregulation of the selective autophagic target p62. Simvastatin induced the activation of AMP-activated protein kinase (AMPK) and its target Raptor, while simultaneously downregulating activation of Akt. Mammalian target of rapamycin (mTOR), a major AMPK/Akt downstream target and a major negative autophagy regulator, and its substrate p70 S6 kinase 1 were also inhibited by simvastatin. Mevalonate, the product of HMG-CoA reductase enzymatic activity, AMPK siRNA or pharmacological inactivation of AMPK with compound C suppressed, while the inhibitors of Akt (10-DEBC hydrochloride) and mTOR (rapamycin) mimicked autophagy induction by simvastatin. Inhibition of autophagy with bafilomycin A1, 3-methyladenine and LC3β shRNA, as well as AMPK inhibition with compound C or AMPK siRNA, markedly increased apoptotic death of simvastatin-treated U251 cells. These data suggest that inhibition of AMPK-dependent autophagic response might sensitize glioma cells to statin-induced apoptotic death.
Galectin‐3 (Gal‐3), an endogenous lectin, exhibits pro‐ and anti‐inflammatory effects in various disease conditions. In order to explore the role of Gal‐3 in NKT‐cell‐dependent pathology, we induced ...hepatitis in C57BL/6 WT and Gal‐3‐deficient mice by using specific ligand for NKT cells: α‐galactosylceramide, glycolipid Ag presented by CD1d. The injection of α‐galactosylceramide significantly enhanced expression of Gal‐3 in liver NKT and dendritic cells (DCs). Genetic deletion or selective inhibition of Gal‐3 (induced by Gal‐3‐inhibitor TD139) abrogated the susceptibility to NKT‐cell‐dependent hepatitis. Blood levels of pro‐inflammatory cytokines (TNF‐α, IFN‐γ, IL‐12) and their production by liver DCs and NKT cells were also downregulated. Genetic deletion or selective inhibition of Gal‐3 alleviated influx of inflammatory CD11c+CD11b+ DCs in the liver and favored tolerogenic phenotype and IL‐10 production of liver NKT and DCs. Deletion of Gal‐3 attenuated the capacity of DCs to support liver damage in the passive transfer experiments and to produce pro‐inflammatory cytokines in vitro. Gal‐3‐deficient DCs failed to optimally stimulate production of pro‐inflammatory cytokines in NKT cells, in vitro and in vivo. In conclusion, Gal‐3 regulates the capacity of DCs to support NKT‐cell‐mediated liver injury, playing an important pro‐inflammatory role in acute liver injury.
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