Maja Misirkic

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.

AMP-activated protein kinase-dependent and -independent mechanisms underlying in vitro antiglioma action of compound C.

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.

Synthesis and in vitro Anticancer Activity of Octahedral Platinum(IV) Complexes with Cyclohexyl‐Functionalized Ethylenediamine‐N,N′‐Diacetate‐Type Ligands

The present study describes the synthesis and anticancer activity of novel octahedral PtIV complexes with cyclohexyl functionalized ethylenediamine‐N,N′‐diacetate‐type ligands. Molecular mechanics calculations and density functional theory analysis revealed that s‐cis is the preferred geometry of these PtIV complexes with tetradentate‐coordinated (S,S)‐ethylenediamine‐N,N′‐di‐2‐(3‐cyclohexyl)propanoate. The viability of cancer cell lines (U251 human glioma, C6 rat glioma, L929 mouse fibrosarcoma, and B16 human melanoma) was assessed by measuring mitochondrial dehydrogenase activity and lactate dehydrogenase release. Cell‐cycle distribution, oxidative stress, caspase activation, and induction of autophagy were analyzed by flow cytometry using appropriate fluorescent reporter dyes. The cytotoxic activity of novel PtIV complexes against various cancer cell lines (IC50 range: 1.9–8.7 μM) was higher than that of cisplatin (IC50 range: 10.9–67.0 μM) and proceeded through completely different mechanisms. Cisplatin induced caspase‐dependent apoptosis associated with the cytoprotective autophagic response. In contrast, the new PtIV complexes caused rapid, caspase‐independent, oxidative stress‐mediated non‐apoptotic cell death characterized by massive cytoplasmic vacuolization, cell membrane damage, and the absence of protective autophagy.

Opposite effects of nanocrystalline fullerene (C60) on tumour cell growth in vitro and in vivo and a possible role of immunosupression in the cancer-promoting activity of C60

In the present study, we compared the effects of nanocrystalline fullerene suspension (nanoC(60)) on tumour cell growth in vitro and in vivo. NanoC(60) suspension was prepared by solvent exchange using tetrahydrofuran to dissolve C(60). In vitro, nanoC(60) caused oxidative stress, mitochondrial depolarization and caspase activation, leading to apoptotic and necrotic death in mouse B16 melanoma cells. Biodistribution studies demonstrated that intraperitoneally injected radiolabeled (125I) nanoC(60) readily accumulated in the tumour tissue of mice subcutaneously inoculated with B16 cells. However, intraperitoneal administration of nanoC(60) over the course of two weeks starting from melanoma cell implantation not only failed to reduce, but significantly augmented tumour growth. The tumour-promoting effect of nanoC(60) was accompanied by a significant increase in splenocyte production of the immunoregulatory free radical nitric oxide (NO), as well as by a reduction in splenocyte proliferative responses to T- and B-cell mitogens ConcanavalinA and bacterial lipopolysaccharide, respectively. A negative correlation between NO production and splenocyte proliferation indicated a possible role of NO in reducing the proliferation of splenocytes from nanoC(60)-injected mice. These data demonstrate that nanoC(60), in contrast to its potent anticancer activity in vitro, can potentiate tumour growth in vivo, possibly by causing NO-dependent suppression of anticancer immune response.

The protection of cells from nitric oxide-mediated apoptotic death by mechanochemically synthesized fullerene (C60) 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.

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.

Modulation of Tumor Necrosis Factor-mediated Cell Death by Fullerenes

PurposeThe fullerene (C60/C70 mixture—C60/70) nanocrystalline suspension prepared by solvent exchange method using tetrahydrofyran (THF/nC60/70) and polyhydroxylated C60/70 [C60/70(OH)n] were compared for their ability to modulate cytotoxicity of the proinflammatory cytokine tumor necrosis factor (TNF).Materials and MethodsTNF-induced cytotoxicity was assessed in L929 fibrosarcoma cells by crystal violet assay. The type of cell death (apoptosis/necrosis), production of reactive oxygen species, mitochondrial depolarization and caspase activation were determined by flow cytometry using the appropriate reporter dyes.ResultsTHF/nC60/70 augmented, while C60/70(OH)n reduced the cytotoxicity of TNF. The numbers of cells undergoing apoptosis/necrosis, as well as of those displaying the activation of apoptosis-inducing enzymes of caspase family, were respectively increased or reduced by THF/nC60/70 or C60/70(OH)n. The antioxidant N-acetylcysteine and mitochondrial permeability transition inhibitor cyclosporin A each partly blocked the cytotoxic action of TNF, indicating the involvement of oxidative stress and mitochondrial dysfunction in the TNF cytotoxicity. Accordingly, THF/nC60/70 or C60/70(OH)n potentiated or suppressed, respectively, TNF-triggered oxidative stress and mitochondrial depolarization.ConclusionThe ability of different fullerene preparations to modulate TNF-induced oxidative stress and subsequent cell death suggests their potential value in the TNF-based cancer therapy or prevention of TNF-dependent tissue damage.

Immunomodulatory actions of central ghrelin in diet-induced energy imbalance

We investigated the effects of centrally administered orexigenic hormone ghrelin on energy imbalance-induced inflammation. Rats were subjected for four weeks to three different dietary regimes: normal (standard food), high-fat (standard food with 30% lard) or food-restricted (70%, 50%, 40% and 40% of the expected food intake in 1st, 2nd, 3rd and 4th week, respectively). Compared to normal-weight controls, starved, but not obese rats had significantly higher levels of proinflammatory cytokines (TNF, IL-1β, IFN-γ) in the blood. When compared to normally fed animals, the hearts of starved and obese animals expressed higher levels of mRNAs encoding proinflammatory mediators (TNF, IL-1β, IL-6, IFN-γ, IL-17, IL-12, iNOS), while mRNA levels of the anti-inflammatory TGF-β remained unchanged. Intracerebroventricular (ICV) injection of ghrelin (1 μg/day) for five consecutive days significantly reduced TNF, IL-1β and IFN-γ levels in the blood of starved rats, as well as TNF, IL-17 and IL-12p40 mRNA expression in the hearts of obese rats. Conversely, ICV ghrelin increased the levels of IFN-γ, IL-17, IL-12p35 and IL-12p40 mRNA in the heart tissue of food-restricted animals. This was associated with an increase of immunosuppressive ACTH/corticosterone production in starved animals and a decrease of the immunostimulatory adipokine leptin both in food-restricted and high-fat groups. Ghrelin activated the energy sensor AMP-activated protein kinase (AMPK) in the hypothalamus and inhibited extracellular signal-regulated kinase (ERK) in the hearts of obese, but not starved rats. Therefore, central ghrelin may play a complex role in energy imbalance-induced inflammation by modulating HPA axis, leptin and AMPK/ERK signaling pathways.

Oxidative stress-mediated hemolytic activity of solvent exchange-prepared fullerene (C60) nanoparticles

The present study investigated the hemolytic properties of fullerene (C(60)) nanoparticles prepared by solvent exchange using tetrahydrofuran (nC(60)THF), or by mechanochemically assisted complexation with macrocyclic oligosaccharide gamma-cyclodextrin (nC(60)CDX) or the copolymer ethylene vinyl acetate-ethylene vinyl versatate (nC(60)EVA-EVV). The spectrophotometrical analysis of hemoglobin release revealed that only nC(60)THF, but not nC(60)CDX or nC(60)EVA-EVV, was able to cause lysis of human erythrocytes in a dose- and time-dependent manner. Atomic force microscopy revealed that nC(60)THF-mediated hemolysis was preceded by erythrocyte shrinkage and increase in cell surface roughness. A flow cytometric analysis confirmed a decrease in erythrocyte size and demonstrated a significant increase in reactive oxygen species production in red blood cells exposed to nC(60)THF. The nC(60)THF-triggered hemolytic activity was efficiently reduced by the antioxidants N-acetylcysteine and butylated hydroxyanisole, as well as by serum albumin, the most abundant protein in human blood plasma. These data indicate that nC(60)THF can cause serum albumin-preventable hemolysis through oxidative stress-mediated damage of the erythrocyte membrane.

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 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. 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, 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. Conclusion: Metformin could reduce food intake by preventing ghrelin-induced AMPK signalling and mTOR inhibition in the hypotalamus.