The Effect of Metformin on Glioma and Melanoma Cell Apoptosis in vitro and Melanoma Growth in vivo

U ovoj disertaciji ispitivan je efekat antidijabetskog leka metformina na apoptozu celijskih linija melanoma i glioma in vitro, u odsustvu i prisustvu hemoterapeutika cisplatina. Takode smo bili zainteresovani za uticaj metformina na rast melanoma in vivo. U odsustvu cisplatina po prvi put je pokazan dvojan antiglioma efekat metformina na celijskoj liniji glioma pacova C6. U celijskim kulturama glioma pacova C6 male gustine metformin je zaustavio progresiju celijskog ciklusa u G0/G1 fazi, bez znacajnog povecanja celijske smrti. Sa druge strane, u konfluentnim kulturama C6 celija metformin je izazvao izuzetno povecanje apoptoze koja je zavisna od kaspaza, depolarizacije mitohondrija, oksidativnog stresa i povezana sa aktivacijom JNK (engl. c-Jun N-terminal kinase). Apoptoza indukovana metforminom je u potpunosti sprecena supstancama koje blokiraju promenu propustljivosti membrane mitohondrija (ciklosporin A) i produkciju reaktivnih kiseonicnih vrsta (N-acetilcistein), dok su je inhibitori aktivacije JNK (SP600125) ili glikolize (natrijumfluorid, jodoacetat) delimicno sprecili. Supstanca C, koja je inhibitor adenozin monofosfatom aktivirane protein kinaze AMPK (engl. AMPactivated protein kinase), je smanjila antiglioma efekat metformina dok je AMPK agonista AICAR (engl. 5-Aminoimidazole-4- carboxamide ribonucleotide) imitirao pomenuti efekat. Primarni astrociti pacova su bili u potpunosti otporni na antiproliferativno i proapoptotsko dejstvo metformina. Dalje je ispitivan in vitro uticaj metformina na antikancersku aktivnost dobro poznatog hemoterapeutika cisplatina. Iako je sam metformin smanjio broj tumorskih celija, iznenadujuce antagonizovao je citotoksicnost cisplatina u U251 celijama glioma. Metformin je smanjio apoptotsku celijsku smrt indukovanu cisplatinom u U251 celijama glioma, tako što je smanjio oksidativni stres i aktivaciju kaspaza. Zapažena citoprotekcija je ocigledno nezavisna od AMPK, jer metformin nije dalje povecao aktivaciju AMPK indukovanu cisplatinom i drugi farmakološki aktivatori AMPK nisu uspeli da inhibiraju apoptozu indukovanu cisplatinom...We investigated the effect of the well known antidiabetic drug metformin on the viability of melanoma and glioma cell lines in vitro, in the absence and presence of cisplatin. Also, we were interested in the effect of metformin on melanoma growth in vivo. In the absence of cisplatin on rat glioma cell line C6, we have shown for the first time a dual antiglioma effect of metformin. In low-density cultures of the C6 rat glioma cell line, metformin blocked the cell cycle progression in G0/G1 phase without inducing significant cell death. In confluent C6 cultures, on the otherhand, metformin caused massive induction of caspase dependent apoptosis associated with c-JunNterminalkinase (JNK) activation, mitochondrial depolarization and oxidative stress. Metformin-triggered apoptosis was completely prevented by agents that block mitochondrial permeability transition (cyclosporin A) and oxygen radical production (N-acetylcisteine), while the inhibitors of JNK activation (SP600125) or glycolysis (sodium fluoride, iodoacetate) provided partial protection. The antiglioma effect of metformin was reduced by compound C, an inhibitor of AMP activated protein kinase (AMPK), and was mimicked by the AMPK agonist AICAR. Rat primary astrocytes were completely resistant to the antiproliferative and proapoptotic action of metformin. Further, it was investigated the influence of metformin on the in vitro anticancer activity of the well-known chemotherapeutic agent cisplatin. Although metformin reduced the number of tumour cells when applied alone, it surprisingly antagonized the cytotoxicity of cisplatin towards U251 human glioma. In U251 glioma cells metformin suppressed cisplatin-induced apoptotic cell death through inhibition of oxidative stress and caspase activation. The observed cytoprotection was apparently AMPKindependent, as metformin did not further increase cisplatin-induced AMPK activation in U251 cells and other pharmacological AMPK activators failed to block cisplatinmediated apoptosis. On the other hand, metformin induced Akt activation in cisplatintreated cells and Akt inhibitor 10-DEBC hydrochloride or phosphoinositide 3- kinase/Akt inhibitor LY294002 abolished metformin-mediated antioxidant and antiapoptotic effects..

Autophagy receptor p62 regulates SARS-CoV-2-induced inflammation in COVID-19

Introduction: Since the interaction between autophagy and virus-induced inflammation is complex, we investigated the interplay between autophagy and inflammation in COVID-19 patients and THP-1 cells expressing SARS-Cov2 proteins NSP5 and ORF3a. Methods: Autophagy markers in blood from 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 level of p62 in cells and its concentration in cell culture supernatants was measured by immunoblot/ELISA. The mRNA levels of proinflammatory cytokines were measured by RT-qPCR. Results: IFN-α, TNF, IL-6, IL-8, IL-17, IL-33, and IFN-γ were elevated in COVID-19 patients at both time points, whereas IL-10 and IL-1β were elevated at admission and one week later, respectively. Autophagy markers LC3 and ATG5 were unchanged in COVID-19. 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 cells caused an autophagy-independent decrease/autophagy-inhibition-dependent increase of intracellular and secreted p62. 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, while a p62 increase in autophagy-deficient cells mirrored the immunostimulatory action of ORF3a. Conclusion: The autophagy receptor p62 is reduced in acute COVID-19, and the balance between autophagy-independent decrease and autophagy blockade-dependent increase of p62 levels could affect SARS-CoV-induced inflammation

The Effect of Metformin on Glioma and Melanoma Cell Apoptosis in vitro and Melanoma Growth in vivo

U ovoj disertaciji ispitivan je efekat antidijabetskog leka metformina na apoptozu celijskih linija melanoma i glioma in vitro, u odsustvu i prisustvu hemoterapeutika cisplatina. Takode smo bili zainteresovani za uticaj metformina na rast melanoma in vivo. U odsustvu cisplatina po prvi put je pokazan dvojan antiglioma efekat metformina na celijskoj liniji glioma pacova C6. U celijskim kulturama glioma pacova C6 male gustine metformin je zaustavio progresiju celijskog ciklusa u G0/G1 fazi, bez znacajnog povecanja celijske smrti. Sa druge strane, u konfluentnim kulturama C6 celija metformin je izazvao izuzetno povecanje apoptoze koja je zavisna od kaspaza, depolarizacije mitohondrija, oksidativnog stresa i povezana sa aktivacijom JNK (engl. c-Jun N-terminal kinase). Apoptoza indukovana metforminom je u potpunosti sprecena supstancama koje blokiraju promenu propustljivosti membrane mitohondrija (ciklosporin A) i produkciju reaktivnih kiseonicnih vrsta (N-acetilcistein), dok su je inhibitori aktivacije JNK (SP600125) ili glikolize (natrijumfluorid, jodoacetat) delimicno sprecili. Supstanca C, koja je inhibitor adenozin monofosfatom aktivirane protein kinaze AMPK (engl. AMPactivated protein kinase), je smanjila antiglioma efekat metformina dok je AMPK agonista AICAR (engl. 5-Aminoimidazole-4- carboxamide ribonucleotide) imitirao pomenuti efekat. Primarni astrociti pacova su bili u potpunosti otporni na antiproliferativno i proapoptotsko dejstvo metformina. Dalje je ispitivan in vitro uticaj metformina na antikancersku aktivnost dobro poznatog hemoterapeutika cisplatina. Iako je sam metformin smanjio broj tumorskih celija, iznenadujuce antagonizovao je citotoksicnost cisplatina u U251 celijama glioma. Metformin je smanjio apoptotsku celijsku smrt indukovanu cisplatinom u U251 celijama glioma, tako što je smanjio oksidativni stres i aktivaciju kaspaza. Zapažena citoprotekcija je ocigledno nezavisna od AMPK, jer metformin nije dalje povecao aktivaciju AMPK indukovanu cisplatinom i drugi farmakološki aktivatori AMPK nisu uspeli da inhibiraju apoptozu indukovanu cisplatinom...We investigated the effect of the well known antidiabetic drug metformin on the viability of melanoma and glioma cell lines in vitro, in the absence and presence of cisplatin. Also, we were interested in the effect of metformin on melanoma growth in vivo. In the absence of cisplatin on rat glioma cell line C6, we have shown for the first time a dual antiglioma effect of metformin. In low-density cultures of the C6 rat glioma cell line, metformin blocked the cell cycle progression in G0/G1 phase without inducing significant cell death. In confluent C6 cultures, on the otherhand, metformin caused massive induction of caspase dependent apoptosis associated with c-JunNterminalkinase (JNK) activation, mitochondrial depolarization and oxidative stress. Metformin-triggered apoptosis was completely prevented by agents that block mitochondrial permeability transition (cyclosporin A) and oxygen radical production (N-acetylcisteine), while the inhibitors of JNK activation (SP600125) or glycolysis (sodium fluoride, iodoacetate) provided partial protection. The antiglioma effect of metformin was reduced by compound C, an inhibitor of AMP activated protein kinase (AMPK), and was mimicked by the AMPK agonist AICAR. Rat primary astrocytes were completely resistant to the antiproliferative and proapoptotic action of metformin. Further, it was investigated the influence of metformin on the in vitro anticancer activity of the well-known chemotherapeutic agent cisplatin. Although metformin reduced the number of tumour cells when applied alone, it surprisingly antagonized the cytotoxicity of cisplatin towards U251 human glioma. In U251 glioma cells metformin suppressed cisplatin-induced apoptotic cell death through inhibition of oxidative stress and caspase activation. The observed cytoprotection was apparently AMPKindependent, as metformin did not further increase cisplatin-induced AMPK activation in U251 cells and other pharmacological AMPK activators failed to block cisplatinmediated apoptosis. On the other hand, metformin induced Akt activation in cisplatintreated cells and Akt inhibitor 10-DEBC hydrochloride or phosphoinositide 3- kinase/Akt inhibitor LY294002 abolished metformin-mediated antioxidant and antiapoptotic effects..

Coordinated time-dependent modulation of AMPK/Akt/mTOR signaling and autophagy controls osteogenic differentiation of human mesenchymal stem cells

We investigated the role of AMP-activated protein kinase (AMPK), Akt, mammalian target of rapamycin (mTOR), autophagy and their interplay in osteogenic differentiation of human dental pulp mesenchymal stem cells. The activation of various members of AMPK, Akt and mTOR signaling pathways and autophagy was analyzed by immunoblotting, while osteogenic differentiation was assessed by alkaline phosphatase staining and real-time RT-PCR/immunoblot quantification of osteocalcin, Runt-related transcription factor 2 and bone morphogenetic protein 2 mRNA and/or protein levels. Osteogenic differentiation of mesenchymal stem cells was associated with early (day 1) activation of AMPK and its target Raptor, coinciding with the inhibition of mTOR and its substrate p70S6 kinase. The early induction of autophagy was demonstrated by accumulation of autophagosome-bound LC3-11, upregulation of proautophagic,beclin-1 and a decrease in the selective autophagic target p62. This was followed by the late activation of Akt/mTOR at days 3-7 of differentiation. The RNA interference-mediated silencing of AMPK, mTOR or autophagy-essential LC3 beta, as well as the pharmacological inhibitors of AMPK (compound C), Akt (10-DEBC hydrochloride), mTOR (rapamycin) and autophagy (bafilomycin A1, chloroquine and ammonium chloride), each suppressed mesenchymal stem cell differentiation to osteoblasts. AMPK knockdown prevented early mTOR inhibition and autophagy induction, as well as late activation of Akt/mTOR signaling, while Ala inhibition suppressed mTOR activation without affecting AMPK phosphorylation. Our data indicate that AMPK controls osteogenic differentiation of human mesenchymal stem cells through both early mTOR inhibition-mediated autophagy and late activation of Akt/mTOR signaling axis

Comparative analysis of cell death mechanisms induced by lysosomal autophagy inhibitors.

We performed a comparative analysis of molecular cytotoxic mechanisms of lysosomal autophagy inhibitors bafilomycin A1, chloroquine, and ammonium chloride in B16 mouse melanoma cells. All agents caused oxidative stress, mitochondrial depolarization, and caspase-dependent apoptotic death, which was not affected by genetic inactivation of autophagy. Cathepsin inhibition reduced only the cytotoxicity of chloroquine, indicating its ability to cause lysosomal membrane permeabilization. Bafilomycin reduced the mRNA levels of anti-apoptotic Bcl-2, while chloroquine and ammonium chloride increased the mRNA expression of pro-apoptotic Pten and Puma, as well as anti-apoptotic Bcl-xL. Ammonium chloride additionally increased the mRNA expression of pro-apoptotic Bim and p53. All three agents decreased the activity of mechanistic target of rapamycin (mTOR) and increased the activation of p38 mitogen-activated protein kinase (MAPK). Chloroquine and ammonium chloride additionally stimulated the phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), respectively, while only bafilomycin increased the phosphorylation of the energy sensor AMP-activated protein kinase (AMPK). mTOR activator leucine did not affect the cytotoxicity of lysosomal inhibitors. p38 MAPK inhibitor SB203580 reduced the cytotoxicity of bafilomycin but increased that of chloroquine and ammonium chloride. The pharmacological inhibition of ERK1/2, JNK, and AMPK potentiated the cytotoxicity of chloroquine, ammonium chloride, and bafilomycin, respectively. The observed mechanistic differences were associated with antagonistic interactions of lysosomal inhibitors in B16 cell killing. In conclusion, all investigated lysosomal inhibitors cause autophagy-independent mitochondrial dysfunction and apoptotic death, but differ in the ability to affect lysosomal permeabilization, balance between pro- and anti-apoptotic molecules of Bcl-2 family, and MAPK/AMPK signaling

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

The protection of cells from nitric oxide-mediated apoptotic death by mechanochemically synthesized fullerene (C-60) nanoparticles

The influence of fullerene (C-60) nanoparticles on the cytotoxicity of a highly reactive free radical nitric oxide (NO) was investigated. Fullerene nanoparticles were prepared by mechanochemically assisted complexation with anionic surfactant sodium dodecyl sulfate, macrocyclic oligosaccharide gamma-cyclodextrin or the copolymer ethylene vinyl acetate-ethylene vinyl versatate. C-60 nanoparticles were characterized by UV-vis and atomic force microscopy. While readily internalized by mouse L929 fibroblasts, C-60 nanoparticles were not cytotoxic. Moreover, they partially protected L929 cells from the cytotoxic effect of NO-releasing compounds sodium nitroprusside (SNP), S-nitroso-N-acetylpenicillamine (SNAP), S-nitrosoglutathione (GSNO) and 3-morpholino-sydnonimine (SIN-1). C-60 nanoparticles reduced SNP-induced apoptotic cell death by preventing mitochondrial depolarization, caspase activation, cell membrane phosphatidylserine exposure and DNA fragmentation. The protective action of C-60 nanoparticles was not exerted via direct interaction with NO, but through neutralization of mitochondria-produced superoxide radical in NO-treated cells, as demonstrated by using different redox-sensitive reporter fluorochromes. These data suggest that C-60 complexes with appropriate host molecules might be plausible candidates for preventing NO-mediated cell injury in inflammatory/autoimmune disorders. (C) 2009 Elsevier Ltd. All rights reserved

Autophagy-independent increase of ATG5 expression in T cells of multiple sclerosis patients.

Autophagy, a process of controlled self-digestion which regulates cell homeostasis, is involved in innate and adaptive immunity. We investigated the expression of autophagy genes and autophagic activity in distinct lymphocyte populations in treatment-naive MS patients. The mRNA and protein levels of autophagy-related (ATG)5, required for autophagosome formation, were increased in CD4+and CD4-T cells, but not B cells of MS patients compared to control subjects. The expression of other investigated autophagy genes, as well as the autophagic activity, did not significantly differ between the two groups. ATG5 mRNA levels in CD4+T cells from MS patients were positively correlated with those of the proinflammatory cytokine tumor necrosis factor. These data suggest that autophagy-independent increase in ATG5 expression might be associated with the proinflammatory capacity of T cells in multiple sclerosis

Intracerebroventricular Administration of Metformin Inhibits Ghrelin-Induced Hypothalamic AMP-Kinase Signalling and Food Intake

Background/Aims: The antihyperglycaemic drug metformin reduces food consumption through mechanisms that are not fully elucidated. The present study investigated the effects of intracerebroventricular administration of metformin on food intake and hypothalamic appetite-regulating signalling pathways induced by the orexigenic peptide ghrelin. Methods: Rats were injected intracerebroventricularly with ghrelin (5 mu g), metformin (50, 100 or 200 mu g), 5-amino-imidazole-4-carboxamide 1-beta-D-ribofuranoside (AICAR, 25 mu g) and L-Ieucine (1 mu g) in different combinations. Food intake was monitored during the next 4 h. Hypothalamic activation of AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase (ACC), regulatory-associated protein of mTOR (Raptor), mammalian target of rapamycin (mTOR) and p70 S6 kinase 1 (S6K) after 1 h of treatment was analysed by immunoblotting. Results: Metformin suppressed the increase in food consumption induced by intracerebroventricular ghrelin in a dose-dependent manner. Ghrelin increased phosphorylation of hypothalamic AMPK and its targets ACC and Raptor, which was associated with the reduced phosphorylation of mTOR. The mTOR substrate, 56K, was activated by intracerebroventricular ghrelin despite the inhibition of mTOR. Metformin treatment blocked ghrelin-induced activation of hypothalamic AMPK/ACC/Raptor and restored mTOR activity without affecting 56K phosphorylation. Metformin also reduced food consumption induced by the AMPK activator AICAR while the ghrelin-triggered food intake was inhibited by the mTOR activator L-leucine. Conclusion: Metformin could reduce food intake by preventing ghrelin-induced AMPK signalling and mTOR inhibition in the hypotalamus. Copyright (c) 2012 S. Karger AG, BaselMinistry of Science and Technological Development of the Republic of Serbia [41025, 175067

Intracerebroventricular Administration of Metformin Inhibits Ghrelin-Induced Hypothalamic AMP-Kinase Signalling and Food Intake

Background/Aims: The antihyperglycaemic drug metformin reduces food consumption through mechanisms that are not fully elucidated. The present study investigated the effects of intracerebroventricular administration of metformin on food intake and hypothalamic appetite-regulating signalling pathways induced by the orexigenic peptide ghrelin. Methods: Rats were injected intracerebroventricularly with ghrelin (5 mu g), metformin (50, 100 or 200 mu g), 5-amino-imidazole-4-carboxamide 1-beta-D-ribofuranoside (AICAR, 25 mu g) and L-Ieucine (1 mu g) in different combinations. Food intake was monitored during the next 4 h. Hypothalamic activation of AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase (ACC), regulatory-associated protein of mTOR (Raptor), mammalian target of rapamycin (mTOR) and p70 S6 kinase 1 (S6K) after 1 h of treatment was analysed by immunoblotting. Results: Metformin suppressed the increase in food consumption induced by intracerebroventricular ghrelin in a dose-dependent manner. Ghrelin increased phosphorylation of hypothalamic AMPK and its targets ACC and Raptor, which was associated with the reduced phosphorylation of mTOR. The mTOR substrate, 56K, was activated by intracerebroventricular ghrelin despite the inhibition of mTOR. Metformin treatment blocked ghrelin-induced activation of hypothalamic AMPK/ACC/Raptor and restored mTOR activity without affecting 56K phosphorylation. Metformin also reduced food consumption induced by the AMPK activator AICAR while the ghrelin-triggered food intake was inhibited by the mTOR activator L-leucine. Conclusion: Metformin could reduce food intake by preventing ghrelin-induced AMPK signalling and mTOR inhibition in the hypotalamus. Copyright (c) 2012 S. Karger AG, BaselMinistry of Science and Technological Development of the Republic of Serbia [41025, 175067