In vitro and in vivo anti-melanoma action of metformin Janjetovic, Kristina; Harhaji-Trajkovic, Ljubica; Misirkic-Marjanovic, Maja ...
European journal of pharmacology,
10/2011, Letnik:
668, Številka:
3
Journal Article
Recenzirano
The in vitro and in vivo anti-melanoma effect of antidiabetic drug metformin was investigated using B16 mouse melanoma cell line. Metformin caused a G
2/M cell cycle arrest associated with apoptotic ...death of melanoma cells, as confirmed by the flow cytometric analysis of cell cycle/DNA fragmentation, phosphatidylserine exposure and caspase activation. Metformin-mediated apoptosis of melanoma cells was preceded by induction of oxidative stress and mitochondrial membrane depolarization, measured by flow cytometry in cells stained with appropriate fluorescent reporter dyes. The expression of tumor suppressor protein p53 was increased, while the mRNA levels of anti-apoptotic Bcl-2 were reduced by metformin, as revealed by cell-based ELISA and real-time RT-PCR, respectively. Treatment with metformin did not stimulate expression of the cycle blocker p21, indicating that p21 was dispensable for the observed cell cycle arrest. The activation of AMP-activated protein kinase (AMPK) was not required for the anti-melanoma action of metformin, as AMPK inhibitor compound C completely failed to restore viability of metformin-treated B16 cells. Metformin induced autophagy in B16 cells, as demonstrated by flow cytometry-detected increase in intracellular acidification and immunoblot-confirmed upregulation of autophagosome-associated LC3-II. Autophagy inhibitors ammonium chloride and wortmannin partly restored the viability of metformin-treated melanoma cells. Finally, oral administration of metformin led to a significant reduction in tumor size in a B16 mouse melanoma model. These data suggest that anti-melanoma effects of metformin are mediated through p21- and AMPK-independent cell cycle arrest, apoptosis and autophagy associated with p53/Bcl-2 modulation, mitochondrial damage and oxidative stress.
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We investigated the effect of large (40 nm) graphene quantum dots (GQDs) in concanavalin A (Con A; 12 mg/kg i.v.)-induced mouse hepatitis, a T cell-mediated liver injury resembling fulminant ...hepatitis in humans. Intravenously injected GQDs (50 mg/kg) accumulated in liver and reduced Con A-mediated liver damage, as demonstrated by histopathological analysis and a decrease in liver lipid peroxidation and serum levels of liver transaminases. The cleavage of apoptotic markers caspase-3/PARP and mRNA levels of proapoptotic mediators Puma, Noxa, Bax, Bak1, Bim, Apaf1, and p21, as well as LC3-I conversion to autophagosome-associated LC3-II and expression of autophagy-related (Atg) genes Atg4b, Atg7, Atg12, and beclin-1, were attenuated by GQDs, indicating a decrease in both apoptosis and autophagy in the liver tissue. This was associated with the reduced liver infiltration of immune cells, particularly the T cells producing proinflammatory cytokine IFN-γ, and a decrease in IFN-γ serum levels. In the spleen of GQD-exposed mice, mRNA expression of IFN-γ and its transcription factor T-bet was reduced, while that of the IL-33 ligand ST2 was increased. The hepatoprotective effect of GQDs was less pronounced in ST2-deficient mice, indicating that it might depend on ST2 upregulation. In vitro, GQDs inhibited splenocyte IFN-γ production, reduced the activation of extracellular signal-regulated kinase in macrophage and T cell lines, inhibited macrophage production of the free radical nitric oxide, and reduced its cytotoxicity toward hepatocyte cell line HepG2. Therefore, GQDs alleviate immune-mediated fulminant hepatitis by interfering with T cell and macrophage activation and possibly by exerting a direct hepatoprotective effect.
Background and Aims:
Galectin-3 Gal-3 is an endogenous lectin with a broad spectrum of immunoregulatory effects: it plays an important role in autoimmune/inflammatory and malignant diseases, but the ...precise role of Gal-3 in pathogenesis of ulcerative colitis is still unknown.
Methods:
We used a model of dextran sulphate sodium DSS-induced acute colitis. The role of Gal-3 in pathogenesis of this disease was tested by evaluating disease development in Gal-3 deficient mice and administration of Gal-3 inhibitor. Disease was monitored by clinical, histological, histochemical, and immunophenotypic investigations. Adoptive transfer was used to detect cellular events in pathogenesis.
Results:
Genetic deletion or pharmacological inhibition of Gal-3 significantly attenuate DSS-induced colitis. Gal-3 deletion suppresses production of pro-inflammatory cytokines in colonic macrophages and favours their alternative activation, as well as significantly reducing activation of NOD-like receptor family, pyrin domain containing 3 NLRP3 inflammasome in macrophages. Peritoneal macrophages isolated from untreated Gal-3-/- mice and treated in vitro with bacterial lipopolysaccharide or DSS produce lower amounts of tumour necrosis factor alpha TNF-α and interleukin beta IL-1β when compared with wild type WT cells. Genetic deletion of Gal-3 did not directly affect total neutrophils, inflammatory dendritic cells DCs or natural killer NK T cells. However, the total number of CD11c+ CD80+ DCs which produce pro-inflammatory cytokines, as well as TNF-α and IL-1β producing CD45+ CD11c- Ly6G+ neutrophils were significantly lower in colons of Gal-3-/- DSS-treated mice. Adoptive transfer of WT macrophages significantly enhanced the severity of disease in Gal-3-/- mice.
Conclusions:
Gal-3 expression promotes acute DSS-induced colitis and plays an important pro-inflammatory role in the induction phase of colitis by promoting the activation of NLRP3 inflammasome and production of IL-1β in macrophages.
The effects of mesenchymal stem cells (MSCs) on the phenotype and function of natural killer T (NKT) cells is not understood. We used concanavalin A (Con A) and α‐galactosylceramide ...(α‐GalCer)‐induced liver injury to evaluate the effects of MSCs on NKT‐dependent hepatotoxicity. Mouse MSCs (mMSCs) significantly reduced Con A‐ and α‐GalCer‐mediated hepatitis in C57Bl/6 mice, as demonstrated by histopathological and biochemical analysis, attenuated the influx of inflammatory T‐bet+, tumour necrosis factor‐α (TNF‐α), interferon‐γ (IFN‐γ)‐producing and GATA3+, interleukin‐4 (IL‐4)‐producing liver NKT cells and downregulated TNF‐α, IFN‐γ and IL‐4 levels in the sera. The liver NKT cells cultured in vitro with mMSCs produced lower amounts of inflammatory cytokines (TNF‐α, IFN‐γ, IL‐4) and higher amounts of immunosuppressive IL‐10 upon α‐GalCer stimulation. mMSC treatment attenuated expression of apoptosis‐inducing ligands on liver NKT cells and suppressed the expression of pro‐apoptotic genes in the livers of α‐GalCer‐treated mice. mMSCs reduced the cytotoxicity of liver NKT cells against hepatocytes in vitro. The presence of 1‐methyl‐dl‐tryptophan, a specific inhibitor of indoleamine 2,3‐dioxygenase (IDO), or l‐NG‐monomethyl arginine citrate, a specific inhibitor of inducible nitric oxide synthase (iNOS), in mMSC‐conditioned medium injected into α‐GalCer‐treated mice, counteracted the hepatoprotective effect of mMSCs in vivo and restored pro‐inflammatory cytokine production and cytotoxicity of NKT cells in vitro. Human MSCs attenuated the production of inflammatory cytokines in α‐GalCer‐stimulated human peripheral blood mononuclear cells in an iNOS‐ and IDO‐dependent manner and reduced their cytotoxicity against HepG2 cells. In conclusion, MSCs protect from acute liver injury by attenuating the cytotoxicity and capacity of liver NKT cells to produce inflammatory cytokines in an iNOS‐ and IDO‐dependent manner.
We explored the interplay between the intracellular energy sensor AMP‐activated protein kinase (AMPK), extracellular signal‐regulated kinase (ERK), and autophagy in phorbol myristate acetate ...(PMA)‐induced neuronal differentiation of SH‐SY5Y human neuroblastoma cells. PMA‐triggered expression of neuronal markers (dopamine transporter, microtubule‐associated protein 2, β‐tubulin) was associated with an autophagic response, measured by the conversion of microtubule‐associated protein light chain 3 (LC3)‐I to autophagosome‐bound LC3‐II, increase in autophagic flux, and expression of autophagy‐related (Atg) proteins Atg7 and beclin‐1. This coincided with the transient activation of AMPK and sustained activation of ERK. Pharmacological inhibition or RNA interference‐mediated silencing of AMPK suppressed PMA‐induced expression of neuronal markers, as well as ERK activation and autophagy. A selective pharmacological blockade of ERK prevented PMA‐induced neuronal differentiation and autophagy induction without affecting AMPK phosphorylation. Conversely, the inhibition of autophagy downstream of AMPK/ERK, either by pharmacological agents or LC3 knockdown, promoted the expression of neuronal markers, thus indicating a role of autophagy in the suppression of PMA‐induced differentiation of SH‐SY5Y cells. Therefore, PMA‐induced neuronal differentiation of SH‐SY5Y cells depends on a complex interplay between AMPK, ERK, and autophagy, in which the stimulatory effects of AMPK/ERK signaling are counteracted by the coinciding autophagic response.
Phorbol myristate acetate (PMA) induces the expression of dopamine transporter, microtubule‐associated protein 2, and β‐tubulin, and subsequent neuronal differentiation of SH‐SY5Y neuroblastoma cells through AMP‐activated protein kinase (AMPK)‐dependent activation of extracellular signal‐regulated kinase (ERK). The activation of AMPK/ERK axis also induces the expression of beclin‐1 and Atg7, and increases LC3 conversion, thereby triggering the autophagic response that counteracts differentiation process.
Phorbol myristate acetate (PMA) induces the expression of dopamine transporter, microtubule‐associated protein 2, and β‐tubulin, and subsequent neuronal differentiation of SH‐SY5Y neuroblastoma cells through AMP‐activated protein kinase (AMPK)‐dependent activation of extracellular signal‐regulated kinase (ERK). The activation of AMPK/ERK axis also induces the expression of beclin‐1 and Atg7, and increases LC3 conversion, thereby triggering the autophagic response that counteracts differentiation process.
Cover Image for this issue: doi: 10.1111/jnc.12872.
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.
Rats were injected intracerebroventricularly with ghrelin (5 µg), metformin (50, 100 or 200 µg), 5-amino-imidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR, 25 µg) and L-leucine (1 µg) 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.
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, S6K, 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 S6K 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.
Metformin could reduce food intake by preventing ghrelin-induced AMPK signalling and mTOR inhibition in the hypotalamus.
We investigated the effect of compound C, a well-known inhibitor of the intracellular energy sensor AMP-activated protein kinase (AMPK), on proliferation and viability of human U251 and rat C6 glioma ...cell lines. Compound C caused G
2/M cell cycle block, accompanied by apoptotic glioma cell death characterized by caspase activation, phosphatidylserine exposure and DNA fragmentation. The mechanisms underlying the pro-apoptotic action of compound C involved induction of oxidative stress and downregulation of antiapoptotic molecule Bcl-2, while no alteration of pro-apoptotic Bax was observed. Compound C diminished AMPK phosphorylation and enzymatic activity, resulting in reduced phosphorylation of its target acetyl CoA carboxylase. AMPK activators metformin and AICAR partly prevented the cell cycle block, oxidative stress and apoptosis induced by compound C. The small interfering RNA (siRNA) targeting of human AMPK mimicked compound C-induced G
2/M cell cycle arrest, but failed to induce oxidative stress and apoptosis in U251 glioma cells. In conclusion, our data indicate that AMPK inhibition is required, but not sufficient for compound C-mediated apoptotic death of glioma cells.
Abstract
We explored the interplay between the intracellular energy sensor
AMP
‐activated protein kinase (
AMPK
), extracellular signal‐regulated kinase (
ERK
), and autophagy in phorbol myristate ...acetate (
PMA
)‐induced neuronal differentiation of
SH
‐
SY
5Y human neuroblastoma cells.
PMA
‐triggered expression of neuronal markers (dopamine transporter, microtubule‐associated protein 2, β‐tubulin) was associated with an autophagic response, measured by the conversion of microtubule‐associated protein light chain 3 (
LC
3)‐I to autophagosome‐bound
LC
3‐
II
, increase in autophagic flux, and expression of autophagy‐related (Atg) proteins Atg7 and beclin‐1. This coincided with the transient activation of
AMPK
and sustained activation of
ERK
. Pharmacological inhibition or
RNA
interference‐mediated silencing of
AMPK
suppressed
PMA
‐induced expression of neuronal markers, as well as
ERK
activation and autophagy. A selective pharmacological blockade of
ERK
prevented
PMA
‐induced neuronal differentiation and autophagy induction without affecting
AMPK
phosphorylation. Conversely, the inhibition of autophagy downstream of
AMPK
/
ERK
, either by pharmacological agents or
LC
3 knockdown, promoted the expression of neuronal markers, thus indicating a role of autophagy in the suppression of
PMA
‐induced differentiation of
SH
‐
SY
5Y cells. Therefore,
PMA
‐induced neuronal differentiation of
SH
‐
SY
5Y cells depends on a complex interplay between
AMPK
,
ERK
, and autophagy, in which the stimulatory effects of
AMPK
/
ERK
signaling are counteracted by the coinciding autophagic response.
image
Phorbol myristate acetate (PMA) induces the expression of dopamine transporter, microtubule‐associated protein 2, and β‐tubulin, and subsequent neuronal differentiation of SH‐SY5Y neuroblastoma cells through AMP‐activated protein kinase (AMPK)‐dependent activation of extracellular signal‐regulated kinase (ERK). The activation of AMPK/ERK axis also induces the expression of beclin‐1 and Atg7, and increases LC3 conversion, thereby triggering the autophagic response that counteracts differentiation process.
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