Vladimir Bumbasirevic

Graphene quantum dots as autophagy-inducing photodynamic agents

The excellent photoluminescent properties of graphene quantum dots (GQD) makes them suitable candidates for biomedical applications, but their cytotoxicity has not been extensively studied. Here we show that electrochemically produced GQD irradiated with blue light (470 nm, 1W) generate reactive oxygen species, including singlet oxygen, and kill U251 human glioma cells by causing oxidative stress. The cell death induced by photoexcited GQD displayed morphological and/or biochemical characteristics of both apoptosis (phosphatidylserine externalization, caspase activation, DNA fragmentation) and autophagy (formation of autophagic vesicles, LC3-I/LC3-II conversion, degradation of autophagic target p62). Moreover, a genetic inactivation of autophagy-essential LC3B protein partly abrogated the photodynamic cytotoxicity of GQD. These data indicate potential usefulness of GQD in photodynamic therapy, but also raise concerns about their possible toxicity.

AMPK-mediated autophagy inhibits apoptosis in cisplatin-treated tumour cells

The role of autophagy in cisplatin anticancer action was investigated using human U251 glioma, rat C6 glioma and mouse L929 fibrosarcoma cell lines. A dose‐ and time‐dependent induction of autophagy was observed in tumour cells following cisplatin treatment, as demonstrated by up‐regulation of autophagy‐inducing protein beclin‐1 and subsequent appearance of acridine orange‐stained acidic autophagic vesicles. The presence of autophagosomes in cisplatin‐treated cells was also confirmed by electron microscopy. Inhibition of autophagy with lysosomal inhibitors bafilomycin A1 and chloroquine, or a PI3 kinase inhibitor wortmannin, markedly augmented cisplatin‐triggered oxidative stress and caspase activation, leading to an increase in DNA fragmentation and apoptotic cell death. The mechanisms underlying the protective effect of autophagy apparently involved the interference with cisplatin‐induced modulation of Bcl‐2 family proteins, as inhibition of autophagy potentiated cisplatin‐mediated up‐regulation of proapoptotic Bax and down‐regulation of anti‐apoptotic Bcl‐2. Autophagy induction in cisplatin‐treated cells was preceded by activation of adenosine monophosphate‐activated protein kinase (AMPK) and concomitant down‐regulation of mammalian target of rapamycin (mTOR)‐mediated phosphorylation of p70S6 kinase. The ability of cisplatin to trigger autophagy was reduced by small interfering RNA (siRNA)‐mediated AMPK silencing, while transfection with mTOR siRNA was sufficient to trigger autophagy in tumour cells. Finally, siRNA‐mediated AMPK down‐regulation and AMPK inhibitor compound C increased cisplatin‐induced tumour cell death, while mTOR siRNA and AMPK activator metformin protected tumour cells from cisplatin. Taken together, these data suggest that cisplatin‐triggered activation of AMPK and subsequent suppression of mTOR activity can induce an autophagic response that protects tumour cells from cisplatin‐mediated apoptotic death.

Metformin reduces cisplatin-mediated apoptotic death of cancer cells through AMPK-independent activation of Akt.

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.

Astrocyte-induced regulatory T cells mitigate CNS autoimmunity.

Although astrocytes presumably participate in maintaining the immune privilege of the central nervous system (CNS), the mechanisms behind their immunoregulatory properties are still largely undefined. In this study, we describe the development of regulatory T cells upon contact with astrocytes. Rat T cells pre‐incubated with astrocytes completely lost the ability to proliferate in response to mitogenic stimuli. The cells were blocked in G0/G1 phase of the cell cycle, expressed less IL‐2R, and produced significantly lower amounts of interferon‐γ (IFN‐γ), but not interleukin‐2 (IL‐2), IL‐10, or tumor necrosis factor (TNF). These anergic cells completely prevented mitogen‐induced growth of normal T lymphocytes, as well as CNS antigen‐driven proliferation of autoreactive T cells. The suppressive activity resided in both CD4+ and CD8+ T‐cell compartments. Heat‐sensitive soluble T‐cell factors, not including transforming growth factor‐β (TGF‐β) or IL‐10, were solely responsible for the observed suppression, as well as for the transfer of suppressive activity to normal T cells. The administration of astrocyte‐induced regulatory T cells markedly alleviated CNS inflammation and clinical symptoms of CNS autoimmunity in rats with experimental allergic encephalomyelitis. Finally, the cells with suppressive properties were readily generated from human lymphocytes after contact with astrocytes. Taken together, these data indicate that astrocyte‐induced regulatory T cells might represent an important mechanism for self‐limitation of excessive inflammation in the brain.

Decreased glutathione levels and altered antioxidant defense in an animal model of schizophrenia: Long-term effects of perinatal phencyclidine administration

Perinatal phencyclidine (PCP) administration to rodents represents one of the more compelling animal models of schizophrenia. There is evidence that decreased glutathione (GSH) levels and oxidative stress mediated through free radicals in the central nervous system are involved in the pathophysiology of this disease. Limited data are available on the role of free radicals in neurotoxicity induced by NMDA-receptor antagonists. The aim of this study was to elucidate the long-term effects of perinatal phencyclidine administration on superoxide dismutase (SOD), catalase (CAT), gamma-glutamyl cisteine ligase (gamma-GCL), glutathione peroxidase (GPx), glutathione reductase (GR) and levels of lipid peroxides as well as GSH content. The Wistar rats were treated on the 2nd, 6th, 9th and 12th postnatal (PN) days with either phencyclidine (10mg/kg) or saline and sacrificed on PN70. The activities of antioxidant enzymes and level of lipid peroxides and GSH were determined in dorsolateral frontal cortex (dlFC), hippocampus, thalamus and caudate nucleus. Expression of SOD1 and SOD2 was determined by immunoblot. Region-specific changes of the measured parameters were observed. Decreased content of reduced GSH and altered activities of GR, GPx and SOD were determined in dlFC. In hippocampus, reduced GSH content and decreased activities of GPx and GR were accompanied with increased activity of gamma-GCL and increased level of lipid peroxides. gamma-GCL and GSH content were also decreased in caudate nucleus, while in thalamus major findings are increased levels of lipid peroxides and GR activity and decreased gamma-GCL activity. It can be concluded that perinatal PCP administration produces long-term alteration of antioxidant defense. Further studies are necessary in order to clarify role of redox dysregulation in the pathogenetic mechanism of schizophrenia.

The prognostic relevance of tumor associated macrophages in advanced stage classical Hodgkin lymphoma

Although the treatment of Hodgkin lymphoma (HL) has been improved, distinguishing reliable prognostic biomarkers could better stratify patients for more effective treatment. We analyzed the prognostic relevance of CD68+ tumor-associated macrophages (TAMs) by immunohistochemical analysis at diagnosis and standard clinical parameters in 52 ABVD (doxorubicin, bleomycin, vinblastine, dacarbazine)-treated patients with advanced stage classical HL (cHL). Patients with >25% CD68+ TAMs compared to those with ≤25% had worse 5-year overall survival (45% vs. 77%, log-rank p = 0.019) and showed a trend toward shorter 5-year event-free survival (51% vs. 71%, log-rank p = 0.19). Additionally, no significant correlation with selected clinical features was found. Significantly shorter 5-year overall survival was associated with International Prognostic Score (IPS) >2, bulky disease, elevated erythrocyte sedimentation rate (log-rank test, p = 0.003, p = 0.049, p = 0.007, respectively). In multivariate analysis, increased CD68+TAMs, IPS >2, and bulky disease were identified as independent prognostic factors for overall survival (Cox multivariate model, p = 0.006, p = 0.007, p = 0.013, respectively). Tumor-associated macrophages represent a potential prognostic biomarker which could contribute to better risk stratification of patients with cHL.

Induction of experimental autoimmune encephalomyelitis in Dark Agouti rats without adjuvant

Experimental autoimmune encephalomyelitis (EAE) is a well‐recognized model for multiple sclerosis (MS) in humans. However, adjuvants used with encephalitogens to induce EAE produce non‐specific effects interfering with the mechanisms involved in the autoimmune response to the central nervous system (CNS) tissue. It is therefore important to establish a more suitable model of EAE for analysis of autoimmune phenomena resembling those operative in MS. Here we report that EAE can be induced regularly in Dark Agouti (DA) strain of rats with spinal cord tissue without any adjuvant, as judged by both clinical and histological parameters. The incidence and severity of EAE depended on the origin of the encephalitogen, the rat versus guinea pig spinal cord homogenate being more efficient. Furthermore, EAE could be reinduced in animals which had recovered from disease that had been induced actively with encephalitogen alone, suggesting the role of adjuvant‐generated non‐specific mechanisms in resistance to reinduction of EAE. Thus, EAE induced in DA rats with encephalitogen alone provides a reproducible model for defining pathogenically relevant events in CNS autoimmunity devoid of the potentially misleading effects of adjuvants.

Chloroquine-mediated lysosomal dysfunction enhances the anticancer effect of nutrient deprivation.

ABSTRACTPurposeTo investigate the ability of chloroquine, a lysosomotropic autophagy inhibitor, to enhance the anticancer effect of nutrient deprivation.MethodsSerum-deprived U251 glioma, B16 melanoma and L929 fibrosarcoma cells were treated with chloroquine in vitro. Cell viability was measured by crystal violet and MTT assay. Oxidative stress, apoptosis/necrosis and intracellular acidification were analyzed by flow cytometry. Cell morphology was examined by light and electron microscopy. Activation of AMP-activated protein kinase (AMPK) and autophagy were monitored by immunoblotting. RNA interference was used for AMPK and LC3b knockdown. The anticancer efficiency of intraperitoneal chloroquine in calorie-restricted mice was assessed using a B16 mouse melanoma model.ResultsChloroquine rapidly killed serum-starved cancer cells in vitro. This effect was not mimicked by autophagy inhibitors or LC3b shRNA, indicating autophagy-independent mechanism. Chloroquine-induced lysosomal accumulation and oxidative stress, leading to mitochondrial depolarization, caspase activation and mixed apoptotic/necrotic cell death, were prevented by lysosomal acidification inhibitor bafilomycin. AMPK downregulation participated in chloroquine action, as AMPK activation reduced, and AMPK shRNA mimicked chloroquine toxicity. Chloroquine inhibited melanoma growth in calorie-restricted mice, causing lysosomal accumulation, mitochondrial disintegration and selective necrosis of tumor cells.ConclusionCombined treatment with chloroquine and calorie restriction might be useful in cancer therapy.

Autophagy-dependent and -independent involvement of AMP-activated protein kinase in 6-hydroxydopamine toxicity to SH-SY5Y neuroblastoma cells.

The role of the main intracellular energy sensor adenosine monophosphate (AMP)-activated protein kinase (AMPK) in the induction of autophagic response and cell death was investigated in SH-SY5Y human neuroblastoma cells exposed to the dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA). The induction of autophagy in SH-SY5Y cells was demonstrated by acridine orange staining of intracellular acidic vesicles, the presence of autophagosome- and autophagolysosome-like vesicles confirmed by transmission electron microscopy, as well as by microtubule-associated protein 1 light-chain 3 (LC3) conversion and p62 degradation detected by immunoblotting. 6-OHDA induced phosphorylation of AMPK and its target Raptor, followed by the dephosphorylation of the major autophagy inhibitor mammalian target of rapamycin (mTOR) and its substrate p70S6 kinase (S6K). 6-OHDA treatment failed to suppress mTOR/S6K phosphorylation and to increase LC3 conversion, p62 degradation and cytoplasmatic acidification in neuroblastoma cells in which AMPK expression was downregulated by RNA interference. Transfection of SH-SY5Y cells with AMPK or LC3β shRNA, as well as treatment with pharmacological autophagy inhibitors suppressed, while mTOR inhibitor rapamycin potentiated 6-OHDA-induced oxidative stress and apoptotic cell death. 6-OHDA induced phosphorylation of p38 mitogen-activated protein (MAP) kinase in an AMPK-dependent manner, and pharmacological inhibition of p38 MAP kinase reduced neurotoxicity, but not AMPK activation and autophagy triggered by 6-OHDA. Finally, the antioxidant N-acetyl cysteine antagonized 6-OHDA-induced activation of AMPK, p38 and autophagy. These data suggest that oxidative stress-mediated AMPK/mTOR-dependent autophagy and AMPK/p38-dependent apoptosis could be valid therapeutic targets for neuroprotection.

Inhibition of AMPK-dependent autophagy enhances in vitro antiglioma effect of simvastatin

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.