Browsing by Author "Pantovic, Aleksandar (36601215300)"

Purpose To investigate the ability of chloroquine, a lysosomotropic autophagy inhibitor, to enhance the anticancer effect of nutrient deprivation. Methods Serum-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 calorierestricted mice was assessed using a B16mouse melanoma model. Results Chloroquine 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 tomitochondrial 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. Conclusion Combined treatment with chloroquine and calorie restriction might be useful in cancer therapy. © Springer Science+Business Media, LLC 2012.

Purpose To investigate the ability of chloroquine, a lysosomotropic autophagy inhibitor, to enhance the anticancer effect of nutrient deprivation. Methods Serum-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 calorierestricted mice was assessed using a B16mouse melanoma model. Results Chloroquine 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 tomitochondrial 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. Conclusion Combined treatment with chloroquine and calorie restriction might be useful in cancer therapy. © Springer Science+Business Media, LLC 2012.

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-II, 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 LC3β, 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 Akt 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. © 2012 Elsevier Inc.

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-II, 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 LC3β, 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 Akt 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. © 2012 Elsevier Inc.

We explored the effect of therapeutic glucoregulation on the blood levels of proinflammatory T helper (Th)17 cytokines interleukin (IL)-17 and IL-23, and Th1 cytokines interferon (IFN)-γ and IL-12 in newly diagnosed type 2 diabetes patients. The investigated group consisted of 23 subjects (17 men and 6 women, age 26-64). The cytokine serum levels, glycated hemoglobin (HbA1c) as a marker of glucoregulation, homeostasis model assessment index as a measure of insulin resistance (HOMA-IR), and body mass index (BMI) were determined before and after 12 weeks of therapy consisting of standard lifestyle modification and metformin (1000. mg b.i.d.). The levels of Th17 and Th1 cytokines before treatment did not correlate with age, BMI or HOMA-IR. The patients with poor glucoregulation (HbA1c. >. 7%, n= 12), compared to those with good glucoregulation (HbA1c. ≤. 7%, n= 11), had higher serum levels of Th17 and Th1 cytokines, but only the differences in IL-17 (median 21.2. pg/ml vs. 4.8. pg/ml) and IFN-γ 5 (0.6. pg/ml vs. 27.7. pg/ml) reached statistical significance (p= 0.003 and p= 0.012, respectively). The reduction of HbA1c values (from 8.6 to 5.9%, p= 0.000) observed upon treatment in patients with poor glucoregulation was associated with a significant decrease in the concentration of IL-17 (from 21.2 to 12.9. pg/ml, p= 0.020), but not IFN-γ (50.6 vs. 52.3, p= 0.349). These data indicate that therapeutic improvement of glucoregulation might contribute to a reduction of IL-17 levels in newly diagnosed type 2 diabetes patients. © 2013 Elsevier GmbH.

We explored the effect of therapeutic glucoregulation on the blood levels of proinflammatory T helper (Th)17 cytokines interleukin (IL)-17 and IL-23, and Th1 cytokines interferon (IFN)-γ and IL-12 in newly diagnosed type 2 diabetes patients. The investigated group consisted of 23 subjects (17 men and 6 women, age 26-64). The cytokine serum levels, glycated hemoglobin (HbA1c) as a marker of glucoregulation, homeostasis model assessment index as a measure of insulin resistance (HOMA-IR), and body mass index (BMI) were determined before and after 12 weeks of therapy consisting of standard lifestyle modification and metformin (1000. mg b.i.d.). The levels of Th17 and Th1 cytokines before treatment did not correlate with age, BMI or HOMA-IR. The patients with poor glucoregulation (HbA1c. >. 7%, n= 12), compared to those with good glucoregulation (HbA1c. ≤. 7%, n= 11), had higher serum levels of Th17 and Th1 cytokines, but only the differences in IL-17 (median 21.2. pg/ml vs. 4.8. pg/ml) and IFN-γ 5 (0.6. pg/ml vs. 27.7. pg/ml) reached statistical significance (p= 0.003 and p= 0.012, respectively). The reduction of HbA1c values (from 8.6 to 5.9%, p= 0.000) observed upon treatment in patients with poor glucoregulation was associated with a significant decrease in the concentration of IL-17 (from 21.2 to 12.9. pg/ml, p= 0.020), but not IFN-γ (50.6 vs. 52.3, p= 0.349). These data indicate that therapeutic improvement of glucoregulation might contribute to a reduction of IL-17 levels in newly diagnosed type 2 diabetes patients. © 2013 Elsevier GmbH.