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In liver the mitochondrial sirtuin, SIRT5, controls ammonia detoxification by regulating CPS1, the first enzyme of the urea cycle. However, while SIRT5 is ubiquitously expressed, urea cycle and CPS1 are only present in the liver and, to a minor extent, in the kidney. To address the possibility that SIRT5 is involved in ammonia production also in nonliver cells, clones of human breast cancer cell lines MDA-MB-231 and mouse myoblast C2C12, overexpressing or silenced for SIRT5 were produced. Our results show that ammonia production increased in SIRT5-silenced and decreased in SIRT5-overexpressing cells. We also obtained the same ammonia increase when using a new specific inhibitor of SIRT5 called MC3482. SIRT5 regulates ammonia production by controlling glutamine metabolism. In fact, in the mitochondria, glutamine is transformed in glutamate by the enzyme glutaminase, a reaction producing ammonia. We found that SIRT5 and glutaminase coimmunoprecipitated and that SIRT5 inhibition resulted in an increased succinylation of glutaminase. We next determined that autophagy and mitophagy were increased by ammonia by measuring autophagic proteolysis of long-lived proteins, increase of autophagy markers MAP1LC3B, GABARAP, and GABARAPL2, mitophagy markers BNIP3 and the PINK1-PARK2 system as well as mitochondrial morphology and dynamics. We observed that autophagy and mitophagy increased in SIRT5-silenced cells and in WT cells treated with MC3482 and decreased in SIRT5-overexpressing cells. Moreover, glutaminase inhibition or glutamine withdrawal completely prevented autophagy. In conclusion we propose that the role of SIRT5 in nonliver cells is to regulate ammonia production and ammonia-induced autophagy by regulating glutamine metabolism.

The following study demonstrated that, in in vitro differentiated neurons, SIRT1 silencing induced an increase of IGF-1 protein expression and secretion and of IGF-1R protein levels which, in turn, prolonged neuronal cell survival in presence of an apoptotic insult. On the contrary, SIRT1 overexpression increased cell death. In particular, IGF-1 and IGF-1R expression levels were negatively regulated by SIRT1. In SIRT1 silenced cells, the increase in IGF-1 and IGF-1R expression was associated to an increase in AKT and ERK1/2 phosphorylation. Moreover, neuronal differentiation was reduced in SIRT1 overexpressing cells and increased in SIRT1 silenced cells. We conclude that SIRT1 silenced neurons appear more committed to differentiation and more resistant to cell death through the activation of IGF-1 survival pathway. J. Cell. Physiol. 228: 1754–1761, 2013. © 2013 Wiley Periodicals, Inc.

Mitochondrial sirtuin 3 (SIRT3) mediates cellular resistance toward various forms of stress. Here, we show that in mammalian cells subjected to hypoxia and staurosporine treatment SIRT3 prevents loss of mitochondrial membrane potential (ΔΨ(mt)), intracellular acidification and reactive oxygen species accumulation. Our results indicate that: (i) SIRT3 inhibits mitochondrial permeability transition and loss of membrane potential by preventing HKII binding to the mitochondria, (ii) SIRT3 increases catalytic activity of the mitochondrial carbonic anhydrase VB, thereby preventing intracellular acidification, Bax activation and apoptotic cell death. In conclusion we propose that, in mammalian cells, SIRT3 has a central role in connecting changes in ΔΨ(mt), intracellular pH and mitochondrial-regulated apoptotic pathways.

A large body of evidence shows that a single bout of strenuous exercise induces oxidative stress in circu- lating human lymphocytes leading to lipid peroxide- tion, DNA damage, mitochondrial perturbations, and protein oxidation. In a training experiment, Wistar rats were divided into control group (CG) and exer- cise group (EG). After a running level exercise until exhaustion, we observed an increase in the mRNA content and protein expression of SIRT1 and SIRT7 in the EG compared to the CG. Moreover, such train- ing exercise did not change mRNA transcripts and protein expression of FOXO3A and GADD45. We also observed an increase of pro-apoptotic protein bax and a decrease of the anti-apoptotic protein bcl-2 in the EG. Accordingly, we observed a caspase-3 activation and poly (ADP-ribose) polymerase (PARP) cleavage only in EG rats. Statistical analysis of the data showed a significant correlation between SIRT1 and SIRT7 expression and apoptotic proteins such as bax, bcl-2 in both tissues. We conclude that, in both muscle, such exercise activates both a damaging apoptotic mecha- nism with bax increase and bcl-2 decrease and a counterbalancing protective mechanism with SIRT1 and SIRT7 increase.

The role of hypoxia in regulating tumor progression is still controversial. Here, we demonstrate that, similarly to what previously observed by us in human prostate and breast tumor samples, hypoxia increases expression of the receptor for advanced glycation end products (RAGE) and the purinergic receptor P2X7 (P2X7R). The role of hypoxia was shown by the fact that hypoxia-inducible factor (HIF)-1α silencing downregulated RAGE and P2X7R protein levels as well as nuclear factor-kappaB (NF-κB) expression. In contrast, NF-κB silencing reduced P2X7R expression without affecting RAGE protein levels or nuclear accumulation of HIF-1α. Treatment of hypoxic tumor cells with HMGB1 and BzATP ligands, respectively, of RAGE and P2X7R, activated a signaling pathway that, through Akt and Erk phosphorylation, determines nuclear accumulation of NF-κB and increases cell invasion. Inhibition of Akt by SH5 and Erk by INH1 prevented both nuclear translocation of NF-κB and cell invasion. Moreover, silencing RAGE and P2X7R abolished nuclear accumulation of NF-κB as well as cell invasion without affecting HIF-1α stabilization. Once in the nucleus, NF-κB would contribute to cell survival and invasion under hypoxia, by maintaining RAGE and P2X7R expression levels and matrix metalloproteinases 2 and 9 synthesis. These results show that, hypoxia can upregulate expression levels of membrane receptors that, by binding extracellular molecules eventually released by necrotic cells, contribute to the increased invasiveness of transformed tumor cells. Moreover, these observations strengthen our working hypothesis that upregulation of damage-associated molecular patterns receptors by HIF-1α represents the crucial event bridging hypoxia and inflammation in obtaining the malignant phenotype.

Extracellular signal-regulated kinase (ERK) 1/2 signaling is involved in tumor cell survival through the regulation of Bcl-2 family members. To explore this further and to demonstrate the central role of the mitochondria in the ERK1/2 pathway we used the HeLa cellular model where apoptosis was induced by tumor necrosis factor (TNF) and cycloheximide (CHX). We show that HeLa cells overexpressing ERK-1 displayed resistance to TNF and CHX. HeLa cells overexpressing a kinase-deficient form of ERK-1 (K71R) were more sensitive to TNF and CHX. In the ERK-1 cells, Bad was phosphorylated during TNF + CHX treatment. In the HeLa wt cells and in the K71R clones TNF and CHX decreased Bad phosphorylation. ERK-1 cells treated with TNF and CHX did not release cytochrome c from the mitochondria. By contrast, HeLa wt and K71R clones released cytochrome c. Bax did not translocate to the mitochondria in ERK-1 cells treated with TNF + CHX. Conversely, HeLa wt and K71R clones accumulated Bax in the mitochondria. In the HeLa wt cells and in both ERK-1 transfectants Bid was cleaved and accumulated in the mitochondria. The caspase-8 inhibitor IETD-FMK and the mitochondrial membrane permeabilization inhibitor bongkrekic acid (BK), partially prevented cell death by TNF + CHX. Anisomycin, a c-Jun N-terminal kinases activator, increased TNF-killing. The ERK-1 cells were resistant to TNF and anisomycin, whereas K71R clones resulted more sensitive. Our study demonstrates that in HeLa cells the ERK-1 kinase prevents TNF + CHX apoptosis by regulating the intrinsic mitochondrial pathway through different mechanisms. Inhibition of the intrinsic pathway is sufficient to almost completely prevent cell death.

The aim of this study was to determine whether heat-shock pretreatment exerted a protective effect against sorbitol-induced apoptotic cell death in K562, U937 and HeLa cell lines and whether such protection was associated with a decreased cytochrome c release from mithocondria and a decreased activation of caspase-9 and -3. Following heat-shock pretreatment (42 ± 0.3°C for 1 hr), these cell lines were exposed to sorbitol for 1 hr. Apoptosis was evaluated by DNA fragmentation, whereas caspase-9,-3 activation, cytochrome c release and heat-shock protein70 (HSP70) were assayed by Western Blot. Sorbitol exposure-induced apoptosis in these different cell lines with a marked activation of caspase-9 and caspase-3, whereas heat-shock pretreatment before sorbitol exposure, induced expression of HSP70 and inhibited sorbitol-mediated cytochrome c release and subsequent activation of caspase-9 and caspase-3. Similarly, overexpression of HSP70 in the three cell lines studied prevented caspase-9 cleavage and activation as well as cell death. Furthermore, we showed that the mRNA expression of iNOS decreased during both the heat-shock treatment and heat-shock pretreatment before sorbitol exposure. By contrast, the expression of Cu-Zn superoxide dismutase (SOD) and Mn-SOD proteins increased during heat-shock pretreatment before sorbitol exposure. We conclude that, heat-shock pretreatment protects different cell lines against sorbitol-induced apoptosis through a mechanism that is likely to involve SOD family members. © 2009 UICC

The maturation of epiphyseal chondrocytes is accompanied by dramatic changes in energy metabolism and shifts in proteins concerned with the induction of apoptosis. We evaluated the role of mitochondria in this process by evaluating the membrane potential (Δψm) of chondrocytes of embryonic tibia and the epiphyseal growth plate. We observed that there was a maturation-dependent change in fluorescence, indicating a fall in the Δψm. The level of mitochondrial Bcl-2 was decreased during maturation, while in the same time period there was an obvious increase in Bax levels in the mitochondrial fraction of the terminally differentiated chondrocytes. BclxL, another anti-apoptotic protein, was also robustly expressed in the mitochondrial fraction, but its expression was not dependent on the maturation status of the chondrocytes. We found that caspase-3 was present throughout the growth plate and in hypertrophic cells in culture. We blocked caspase-3 activity and found that alkaline phosphatase staining and mineral formation was decreased, and the cells had lost their characteristic shape. Moreover, we noted that the undifferentiated cells were insensitive to elevated concentrations of inorganic phosphate (Pi). It is concluded that during hypertrophy, the change in membrane potential, the increased binding of a pro-apoptotic protein to mitochondria, and the activation of caspase-3 serve to prime cells for apoptosis. Only when the terminally differentiated chondrocytes are challenged with low levels of apoptogens there is activation of apoptosis. J. Cell. Physiol. 210: 609–615, 2007. © 2006 Wiley-Liss, Inc.

The research of the last decade highlighted the existence of a family of genes activated by cellular stresses that allow the cells to reactivate defense and repair activities regardless of age. The prolonged activation of these genes enhances the organism health and lifespan. Members of this gene family are called sirtuins (SIRT). The founding member of the SIRT protein family, Sir2 is a limiting component of yeast longevity. Many members of this family have been also identified as key longevity regulators in species ranging from yeast to fly. On the other hand, the role of SIRTs in the regulation of mammalian ageing has been questioned. While SIRTs' effects on lifespan are still a matter of scientific debate, the beneficial effects of SIRTs in terms of physical health and quality of aging are widely accepted. Increasing evidence suggests a pivotal role for SIRTs in mediating the adaptive response to physical exercise. The following review summarizes the knowledge so far acquired on sirtuins' role in mediating beneficial effects of physical exercise. In particular, the first paragraph gives an overture on mammalian sirtuins defining their localization, function when possible, and substrates. In the second paragraph, we discuss recent data regarding alteration of sirtuins expression and activity after physical exercise collected by our laboratory and others'.

Myogenic transcription is repressed in myoblasts by serum-activated cyclin-dependent kinases, such as cdk2 and cdk4. Serum withdrawal promotes muscle-specific gene expression at least in part by down-regulating the activity of these cdks. Unlike the other cdks, cdk9 is not serum- or cell cycle-regulated and is instead involved in the regulation of transcriptional elongation by phosphorylating the carboxyl-terminal domain (CTD) of RNA polymerase II. While ectopic expression of cdk2 together with its regulatory subunits (cyclins E and A) inhibits myogenic transcription, overproduction of cdk9 and its associated cyclin (cyclin T2a) strengthens MyoD-dependent transcription and stimulates myogenic differentiation in both MyoD-converted fibroblasts and C2C12 muscle cells. Conversely, inhibition of cdk9 activity by a dominant negative form (cdk9-dn) represses the myogenic program. Cdk9, cyclinT2 and MyoD can be detected in a multimeric complex in C2C12 cells, with the minimal cdk9-binding region of MyoD mapping within 101-161 aa of the bHLH region. Finally, cdk9 can phosphorylate MyoD in vitro, suggesting the possibility that cdk9/cycT2a regulation of muscle differentiation includes the direct enzymatic activity of the kinase on MyoD.

This review summarizes the experimental evidence that supports the role of dopamine in the regulation of ocular axial growth. The most important functions attributed to dopamine are light adaptation and regulation of the retinal circadian rhythm. An increase of the retinal levels of dopamine activates D1 and D2 dopaminergic receptors present throughout the retina, generating a signal that inhibits axial growth once the eye has reached emmetropization. Researchers induced form-deprivation myopia in animal models in order to assess the different changes of ocular axial growth. Other studies have shown that phenylethylamine is an endogenous precursor-neurotransmitter capable of modulating the activity of dopamine. Considering the role of the dopaminergic system in the development of myopia (in children and adolescents) and the fact that phenylethylamine improves the consequences of a dopamine deficit, it would be interesting to study the effect of phenylethylamine on the regulation of axial growth, which represents the genesis of myopia.

Reprogramming technologies have been developed to revert somatic differentiated cells into pluripotent stem cells that can be differentiated into different lineages potentially useful in stem cell therapy. Reprogramming methods have been progressively refined to increase their efficiency, to obtain a cell population suitable for differentiation, and to eliminate viral plasmid which could be responsible for many unwanted side-effects when used in personalized medicine. All these methods are aimed to introduce into the cell genes or mRNAs encoding a set of four transcription factors (OCT- 4, SOX-2, KLF-4 and c-MYC) or a set of three lincRNAs (large intragenic non-coding RNAs) acting downstream of the reprogramming transcription factors OCT-4, SOX-2 and NANOG. Translational clinical applications in human pathologies and in developmental, repair and cancer biology have been numerous. Cancer cells can be, at least in principle, reprogrammed into a normal phenotype. This is a recently raised issue, rapidly advancing in many human tumors, especially endocrine-related cancers, such as breast, prostate and ovarian ca. The present review aims to describe basic phenomena observed in reprogramming tumor cells and solid tumors and to discuss their meaning in human hormone-related cancers. We will also discuss the fact that some of the targeted transcription factors are "normally" activated in a number of physiological processes, such as morphogenesis, hypoxia and wound healing, suggesting an in vivo role of reprogramming for development and homeostasis. Finally, we will review concerns and warnings raised for in vivo reprogramming of human tumors and for the use of induced pluripotent stem cells (iPSCs) in human therapy.

E2F-1-activated transcription promotes cell cycle progression and apoptosis. These functions are regulated by several factors including the E2F-1-binding protein MDM2 and the retinoblastoma protein pRb. Using a yeast two-hybrid screen we have identified the MDM2-related protein, MDMX, as an E2F-1-binding protein. In these studies we find that coexpression of MDMX with E2F-1 results in degradation of the MDMX protein. Although this proteolytic degradation can be blocked by the protease inhibitors bafilomycin A(1), N-acetyl-Leu-Leu-Norleu-AL, and N-acetyl-Leu-Leu-Met-AL, MDMX degradation is not inhibited by lactacystin, suggesting that degradation occurs by a proteasome-independent mechanism. Using an E2F-1 deletion mutant (E2F-1(180-437)) we show that E2F-1-targeted degradation of MDMX does not require the E2F-1 DNA binding domain and therefore is independent of E2F-1-driven transcription. We also find that this transcriptionally inactive E2F-1 mutant is capable of degrading the MDMX-related protein MDM2 and the MDMX isoform MDMX-S. Mapping of the E2F-1 C terminus reveals that neither a previously characterized C-terminal MDM2 binding domain nor the pRb binding domain on E2F-1 is required for MDMX and MDM2 degradation.

The multipotential murine embryonic C3H10T1/2 mesenchymal cell line is able to undergo chondrogenesis in vitro, in a high density micromass environment, following treatment with soluble human bone morphogenetic protein-2 (BMP-2). To enhance this process, the human BMP-2 cDNA was cloned into a retroviral expression vector and a high titer, infectious retrovirus (replication defective) was generated. Infection of C3HIOT1/2 cells with this retroviral construct resulted in an infection efficiency of 90-95% and was highly effective in converting cells in micromass culture to a chondrocyte phenotype, as assessed by positive Alcian blue staining for extracellular matrix proteoglycans, increased sulfate incorporation, increased expression of the cartilage marker genes collagen type II and aggrecan, and decreased expression of collagen type I. Interestingly, BMP-2 expression in the micromass cultures also induced the expression of the cell cycle inhibitory protein/differentiation factor p21/WAF1, suggesting its functional involvement in chondrogenesis. The chondrogenic effect of retrovirally expressed BMP-2 in these high-density cultures was limited to the infected cells, since uninfected cells did not chondrify when co-cultured as a nonoverlapping micromass adjacent to BMP-2 expressing cells. These data indicate that retrovirally expressed BMP-2 is highly effective at inducing a chondrocyte phenotype in a multipotential mesenchymal cell line in vitro, and its action is restricted to the infected cell population. These findings should provide a framework for the optimization of chondrogenesis in culture using mesenchymal stem cells and retroviral gene transfer.

The following study demonstrated that, in in vitro differentiated neurons, SIRT1 silencing induced an increase of IGF-1 protein expression and secretion and of IGF-1R protein levels which, in turn, prolonged neuronal cell survival in presence of an apoptotic insult. On the contrary, SIRT1 overexpression increased cell death. In particular, IGF-1 and IGF-1R expression levels were negatively regulated by SIRT1. In SIRT1 silenced cells, the increase in IGF-1 and IGF-1R expression was associated to an increase in AKT and ERK1/2 phosphorylation. Moreover, neuronal differentiation was reduced in SIRT1 overexpressing cells and increased in SIRT1 silenced cells. We conclude that SIRT1 silenced neurons appear more committed to differentiation and more resistant to cell death through the activation of IGF-1 survival pathway.

In this study we investigate: 1) the role of multidrug resistance protein-4 (MRP4), an organic anion unidirectional transporter, in modulating aspirin action on human platelet cyclooxygenase (COX)-1; and 2) whether the impairment of aspirin-COX-1 interaction, found in coronary artery bypass grafting (CABG) patients, could be dependent on MRP4-mediated transport.Platelets of CABG patients present a reduced sensitivity to aspirin despite in vivo and in vitro drug treatment. Aspirin is an organic anion and could be a substrate for MRP4.Intracellular aspirin concentration and drug COX-1 activity, measured by thrombin-induced thromboxane B2 (TxB2) production, were evaluated in platelets obtained from healthy volunteers (HV) and hematopoietic-progenitor cell cultures reducing or not reducing MRP4-mediated transport. Platelet MRP4 expression was evaluated, in platelets from HV and CABG patients, by dot-blot or by immunogold-electromicrographs or immunofluorescence-microscopy analysis.Inhibition of MRP4-mediated transport by dipyridamole or Mk-571 increases aspirin entrapment and its in vitro effect on COX-1 activity (142.7 ± 34.6 pg/10(8) cells vs. 343.7 ± 169.3 pg/10⁸ cells TxB2-production). Platelets derived from megakaryocytes transfected with MRP4 small interfering ribonucleic acid have a higher aspirin entrapment and drug COX-1 activity. Platelets from CABG patients showed a high expression of MRP4 whose in vitro inhibition enhanced aspirin effect on COX-1 (349 ± 141 pg/10⁸ cells vs. 1,670 ± 646 pg/10⁸ cells TxB2-production).Aspirin is a substrate for MRP4 and can be extruded from platelet through its transportation. Aspirin effect on COX-1 is little-related to MRP4-mediated aspirin transport in HV, but in CABG patients with MRP4 over-expression, its pharmacological inhibition enhances aspirin action in an efficient way.

Background A large body of evidence shows that a single bout of strenuous exercise induces oxidative stress in circulating human lymphocytes leading to lipid peroxidation, DNA damage, mitochondrial perturbations, and protein oxidation. In our research, we investigated the effect of physical load on the extent of apoptosis in primary cells derived from blood samples of sixteen healthy amateur runners after marathon (a.m.). Results Blood samples were collected from ten healthy amateur runners peripheral blood mononuclear cells (PBMCs) were isolated from whole blood and bcl-2, bax, heat shock protein (HSP)70, Cu-Zn superoxide dismutase (SOD), Mn-SOD, inducible nitric oxide synthase (i-NOS), SIRT1, SIRT3 and SIRT4 (Sirtuins) RNA levels were determined by Northern Blot analysis. Strenuous physical load significantly increased HSP70, HSP32, Mn-SOD, Cu-Zn SOD, iNOS, GADD45, bcl-2, forkhead box O (FOXO3A) and SIRT1 expression after the marathon, while decreasing bax, SIRT3 and SIRT4 expression (P < 0.0001). Conclusion These data suggest that the physiological load imposed in amateur runners during marathon attenuates the extent of apoptosis and may interfere with sirtuin expression.

Survival strategies adopted by tumor cells in response to a hypoxic stress include activation of hypoxia-inducible factor 1 (HIF-1) and autophagy. However, the importance and the function of each molecular response is not well defined. In the present study, we investigated invasiveness, migration, matrix metalloproteinases (MMPs) activity, and cell survival of MDA-MB-231 cells under normoxia, hypoxia, and hypoxia/reoxygenation (H/R). Moreover, to assess the importance of hypoxia and autophagy on the parameters studied, cells were either left untreated or treated with Chetomin (a selective inhibitor of HIF-1α) or trifluoperazine (TFP, an activator of autophagy). We found that hypoxia and H/R stimulated invasiveness and migration of MDA-MB-231 cells with an increased MMP-2 activity. Chetomin and TFP differently regulated the cellular behavior under the oxygenation conditions studied. In fact, Chetomin was most effective in inhibiting cell invasion, MMPs activity, and cell survival under hypoxia but not normoxia or H/R. By contrast, TFP inhibition of cell invasion, migration, and cell survival was independent from oxygenation conditions. TFP-induced autophagy was inhibited by light chain protein 3 (LC3) silencing or 3-methyladenine (3MA) treatment. In fact, LC3-silenced cells were able to invade in the presence of TFP without any GATE16 processing and p62 degradation. Immunofluorescence assay showed that LC3 silencing inhibited TFP-induced autophagosome formation. However, we also showed that both TPF treatment and LC3 silencing caused cytoskeleton impairments suggesting a possible interaction between LC3 and cytoskeleton components. In conclusion, our study shows that hypoxia and autophagy by acting on common (HIF-1α) or separate (MMPs, cytoskeleton) targets differently regulate cell invasion, MMPs activity, and survival. J. Cell. Physiol. 223: 359–368, 2010. © 2010 Wiley-Liss, Inc.

The aim of this study was to determine whether heat-shock pretreatment exerted a protective effect against sorbitol-induced apoptotic cell death in K562, U937 and HeLa cell lines and whether such protection was associated with a decreased cytochrome c release from mithocondria and a decreased activation of caspase-9 and -3. Following heat-shock pretreatment (42 +/- 0.3 degrees C for 1 hr), these cell lines were exposed to sorbitol for 1 hr. Apoptosis was evaluated by DNA fragmentation, whereas caspase-9,-3 activation, cytochrome c release and heat-shock protein70 (HSP70) were assayed by Western Blot. Sorbitol exposure-induced apoptosis in these different cell lines with a marked activation of caspase-9 and caspase-3, whereas heat-shock pretreatment before sorbitol exposure, induced expression of HSP70 and inhibited sorbitol-mediated cytochrome c release and subsequent activation of caspase-9 and caspase-3. Similarly, overexpression of HSP70 in the three cell lines studied prevented caspase-9 cleavage and activation as well as cell death. Furthermore, we showed that the mRNA expression of iNOS decreased during both the heat-shock treatment and heat-shock pretreatment before sorbitol exposure. By contrast, the expression of Cu-Zn superoxide dismutase (SOD) and Mn-SOD proteins increased during heat-shock pretreatment before sorbitol exposure. We conclude that, heat-shock pretreatment protects different cell lines against sorbitol-induced apoptosis through a mechanism that is likely to involve SOD family members.

Kaempferol (3,4',5,7-tetrahydroxyflavone) is a flavonoid with anti- and pro-oxidant activity present in various natural sources. Kaempferol has been shown to posses anticancer properties through the induction of the apoptotic program. Here we report that treatment of the chronic myelogenous leukemia cell line K562 and promyelocitic human leukemia U937 with 50 microM kaempferol resulted in an increase of the antioxidant enzymes Mn and Cu/Zn superoxide dismutase (SOD). Kaempferol treatment induced apoptosis by decreasing the expression of Bcl-2 and increasing the expressions of Bax. There were also induction of mitochondrial release of cytochrome c into cytosol and significant activation of caspase-3, and -9 with PARP cleavage. Kaempferol treatment increased the expression and the mitochondria localization of the NAD-dependent deacetylase SIRT3. K562 cells stably overexpressing SIRT3 were more sensitive to kaempferol, whereas SIRT3 silencing did not increase the resistance of K562 cells to kaempferol. Inhibition of PI3K and de-phosphorylation of Akt at Ser473 and Thr308 was also observed after treating both K562 and U937 cells with kaempferol. In conclusion our study shows that the oxidative stress induced by kaempferol in K562 and U937 cell lines causes the inactivation of Akt and the activation of the mitochondrial phase of the apoptotic program with an increase of Bax and SIRT3, decrease of Bcl-2, release of cytochrome c, caspase-3 activation, and cell death.

Adaptation to hypoxia through activation of the hypoxia inducible factor-1 (HIF-1) is crucial for tumor cells survival. Here we describe the antitumoral effects of the new molecule CR 3294 on tumor cells in the presence of hypoxia. Treatment of the breast carcinoma cell line MDA-MB-231 with CR 3294 in 1% O(2) resulted in an in vivo and in vitro inhibition of tumor growth. CR 3294 induced accumulation of autophagosomes in hypoxic MDA-MB-231 cells as assessed by both transmission electron microscopy (TEM) and the autophagic marker LC3-II. TEM analysis revealed the presence of invaginations of the cytoplasm into the nucleus. Autophagosomes were present in such invaginations. Moreover, CR 3294 inhibited both the DNA binding of HIF-1alpha and VEGF mRNA synthesis. Immunoprecipitation and immunofluorescence studies showed an interaction between LC3 and HIF-1alpha. We next detailed the effect of inhibitors and activators of autophagy on both HIF-1alpha and LC3. In particular, 3 methyladenine (3MA) and wortmannin, two macroautophagic inhibitors, prevented both the decrease of HIF-1alpha protein levels and LC3 processing in cells treated with CR 3294. Bafilomycin and leupeptin, inhibitors of lysosomes, prevented HIF-1alpha decrease without affecting LC3 processing. By contrast, treating hypoxic MDA-MB-231 cells with trifluoperazine (TFP) or serum withdrawal (SW), two activators of autophagy, diminished HIF-1alpha levels and stimulated LC3 processing. These results indicate that activation of the autophagic pathway in hypoxic cells by the new molecule CR 3294, as well as by TFP or SW, can have potentially important implications for cancer treatment.

Induction of cell death in HeLa cells with TNF and cycloheximide (CHX) required an adequate ATP supply and was accompanied by decrease in intracellular pH, translocation of Bax, perinuclear clustering of the mitochondria, and cytochrome c release. The chloride channel inhibitor furosemide prevented the intracellular acidification, the translocation of Bax and the cell death. Cyclosporin A (CyA) or bongkrekic acid (BK) inhibited the induction of the MPT, the release of cytochrome c and the cell death without affecting the perinuclear clustering of the mitochondria or the translocation of Bax. Energy depletion with the ATP synthase inhibitor oligomycin or the uncoupler FCCP in the presence of 2-deoxy-glucose prevented the perinuclear clustering of the mitochondria and the cell killing. However, mitochondrial translocation of Bax was still observed. By contrast, cytochrome c was released in the oligomycin-treated cells but not in the same cells treated with FCCP. The data demonstrate that apoptosis in HeLa cells is ATP dependent and requires the translocation of Bax. The movement of Bax to the mitochondria occurs before and during the perinuclear clustering of these organelles and does not require the presence of ATP. The release of cytochrome c depends on the induction of the mitochondrial permeability transition but not ATP content.

NG108-15 cells differentiate into neurons by 1 mM sodium butyrate (NaB) treatment. Differentiated cells resulted more resistant to staurosporine (STS) than proliferating cells. In particular, STS treatment decreased Bcl-2 and Bcl-x(L) content in mitochondria of proliferating cells, but not in mitochondria of differentiated cells. Bad was phosphorylated and downregulated only in differentiated cells. Bax accumulated in the mitochondria of proliferating but not differentiated cells. Mitochondrial release of cytochrome c was observed in proliferating cells, whereas mitochondria of differentiated cells retained cytochrome c. Proliferating cells treated with STS accumulated Endo G and AIF in the nucleus. By contrast, differentiated cells did not show such nuclear accumulation. Treatment of differentiated cells with Insulin-like Growth Factor-1 (IGF-1) and STS resulted in a 17.1% increase of cell viability. The survival role of IGF-1 was demonstrated by treating differentiated cells with an anti-IGF-1 neutralizing antibody. Such treatment significantly increased STS-induced cell death. Electrophysiology studies showed that in STS-treated cells membrane potential oscillations were reduced in amplitude and did not give rise to spontaneous action potentials (APs). However, the percentage of cells yielding overshooting APs returned to control values after STS removal. It is concluded that neuronal differentiation of NG108-15 cells induces resistance to apoptotic cell death and that IGF-1 plays a central role in sustaining this mechanism.

This study shows that in the glioblastoma hamster cell line HJC12 the retinoblastoma family member pRb2/p130 enhances gamma-radiation-induced cell death. In HJC12 cells the tetracycline-regulated expression of pRb2/p130 increased the percentage of gamma-radiation-induced apoptotic cells from 27 to 47%. pRb2/p130 overexpression was associated with the downregulation of the anti-apoptotic factor Bcl-2 and the upregulation of the steady-state protein levels of the pro-apoptotic transcription factor p73. In particular, RT-PCR showed a significant increase in the expression of the p73delta isoform when pRb2/p130 was overexpressed. The ability of pRb2/p130 to modulate apoptosis was not associated with its role in mediating G0/G1 arrest during cell cycle progression. Our data suggest a role for pRb2/p130 in glioblastoma gamma-radiation-induced cell death, indicating that the antitumoral action of pRb2/p130 can regulate both inhibition of cell cycle progression and induction of cell death.

A large body of evidence shows that a single bout of strenuous exercise induces oxidative stress in circulating human lymphocytes leading to lipid peroxidation, DNA damage, mitochondrial perturbations, and protein oxidation.In our research, we investigated the effect of physical load on the extent of apoptosis in primary cells derived from blood samples of sixteen healthy amateur runners after marathon (a.m.).Blood samples were collected from ten healthy amateur runners peripheral blood mononuclear cells (PBMCs) were isolated from whole blood and bcl-2, bax, heat shock protein (HSP)70, Cu-Zn superoxide dismutase (SOD), Mn-SOD, inducible nitric oxide synthase (i-NOS), SIRT1, SIRT3 and SIRT4 (Sirtuins) RNA levels were determined by Northern Blot analysis. Strenuous physical load significantly increased HSP70, HSP32, Mn-SOD, Cu-Zn SOD, iNOS, GADD45, bcl-2, forkhead box O (FOXO3A) and SIRT1 expression after the marathon, while decreasing bax, SIRT3 and SIRT4 expression (P0.0001).These data suggest that the physiological load imposed in amateur runners during marathon attenuates the extent of apoptosis and may interfere with sirtuin expression.

We examined the effect of paclitaxel on human osteoblastic cells Saos-2 to determine if paclitaxel can affect proliferation and apoptosis. We used a p53-negative cell line in order to mimic the loss of function frequently observed at the clinical level. Paclitaxel induced cell death in a dose- and time-dependent manner. Marked nuclear condensation and fragmentation of chromatin were observed by Hoechst 33258 stain, DNA ladder formation, electron microscopy, and flow cytometry at concentrations as low as 100 nM, a concentration which can be achieved by infusion in human plasma. At 100 nM, paclitaxel induced a G2 arrest at 8 h of treatment. The cells then continued to accumulate in G2 until 72 h when the percentage of apoptotic events reached 54%. At the molecular level, Bcl-2 protein was phosphorylated at 16 h and PARP protein was cleaved, indicating the activation of caspase-3-like proteases. Caspase inhibitors Z-VAD-FMK and Z-DEVD-FMK rescued Saos-2 cells from paclitaxel-induced apoptosis. CD95 expression was constantly high, while CD95L showed a threefold increase in expression. This suggests that, following the G2 arrest, apoptosis is induced through the CD95/CD95L system.

Introduction: Molecular chaperones have many functions, such as protecting other proteins against aggregation, assisting in folding of nascent proteins/refolding of damaged proteins and targeting severely damaged proteins to degradation. As one of the molecular chaperones, Hsp90 functions to facilitate the folding of newly synthesized and denatured client proteins, including mutated p53, Bcr-Abl, p185ErbB2 and Raf-1. The Bcr-Abl fusion gene encodes for the p210Bcr-Abl tyrosine kinase (TK) implicated in the pathogenesis of chronic myelogenous leukemia (CML). Studies in cultured cells have identified many signal transduction pathways activated by Bcr-Abl, including activation of the Ras, MAPK, JNK/SAPK, phosphatidylinositol-3 kinase, nuclear factor-B and STAT pathways. Imatinib mesylate (imatinib IM) is a tyrosine kinase inhibitor that competitively inhibits ATP binding in the kinase domains of both the Bcr-Abl and c-Abl kinases. It has been suggested that resistance to imatinib stems from Bcr-Abl gene amplification, leading to overexpression of Bcr-Abl protein or point mutations in the Bcr-Abl gene However, several groups suggested that there might be other forms of Bcr-Abl-independent imatinib resistance Recently, it has been reported that changes in histone deacetylase (HDAC) expression in leukemic cells could be involved in mechanisms for abnormal cellular proliferation that operate through chromatin-independent pathways and thereby could lead to acquired drug resistance of the cells In the present study, we evaluated in primary leukemic blasts, obtained from chronic myelogenous leukemia patients at onset, patients in blast crisis and patients which were imatinib-resistant The espression the sirtuin members family and HSP70, HSP90 i-NOS and bcl-2 was evaluated by Nortern blot and Western blot analysis. Material and Methods: Primary leukaemia blasts We harvested primary blast rich mononuclear cells were obtained by gradient centrifugation on ficoll-hypaque of bone marrow and peripheral blood cells after obtaining appropriate informed consent. Northern blot Total RNAs from control or treated cells were isolated using Tri Reagent Aliquots of RNA were electrophoresed and blotted onto nylon membranes, that hybridized to 32P-labelled probe. Western Blot Cells were lysed and. then were centrifugated. Protein concentration was determined by the Bradford assay.. Equivalent amounts of protein loaded and electrophoresed and were transferred to nitrocellulose membranes, that were incubated with the different primary antibodies:, Result and Discussion:. In the present study, we evaluated a pattern of different gene expression by Northern Blot and Western Blot analysis in bone marrow and peripheral blood cells from 16 CML patients at onset, from 2 patients in blast crisis evolved under IM treatment, and 14 imatinib-resistant patients. Some RNAs were underexpressed in most or all samples tested and never overexpressed (eg SIRT2, SIRT3, SIRT4 and SIRT5), while others were overexpressed in the great majority of samples and rarely, if ever, underexpressed (eg SIRT1, SIRT7, HSP90, iNOS)Furthermore, we examined the level of heat-shock related proteins HSP90 and bcl-2 in 2 patients during treatment with IM. and one patient IM-resistant by western Blot analysis: HSP90 and BCl-2 increased one patient during treatment with IM, while both protein levels was very high in one one patient IM-resistant These results suggest that the difference of genes expression might contribute to patterns of clinical response.

ObjectivesIn this study we investigate: 1) the role of multidrug resistance protein-4 (MRP4), an organic anion unidirectional transporter, in modulating aspirin action on human platelet cyclooxygenase (COX)-1; and 2) whether the impairment of aspirin–COX-1 interaction, found in coronary artery bypass grafting (CABG) patients, could be dependent on MRP4-mediated transport.BackgroundPlatelets of CABG patients present a reduced sensitivity to aspirin despite in vivo and in vitro drug treatment. Aspirin is an organic anion and could be a substrate for MRP4.MethodsIntracellular aspirin concentration and drug COX-1 activity, measured by thrombin-induced thromboxane B2 (TxB2) production, were evaluated in platelets obtained from healthy volunteers (HV) and hematopoietic-progenitor cell cultures reducing or not reducing MRP4-mediated transport. Platelet MRP4 expression was evaluated, in platelets from HV and CABG patients, by dot-blot or by immunogold-electromicrographs or immunofluorescence-microscopy analysis.ResultsInhibition of MRP4-mediated transport by dipyridamole or Mk-571 increases aspirin entrapment and its in vitro effect on COX-1 activity (142.7 ± 34.6 pg/108 cells vs. 343.7 ± 169.3 pg/108 cells TxB2-production). Platelets derived from megakaryocytes transfected with MRP4 small interfering ribonucleic acid have a higher aspirin entrapment and drug COX-1 activity. Platelets from CABG patients showed a high expression of MRP4 whose in vitro inhibition enhanced aspirin effect on COX-1 (349 ± 141 pg/108 cells vs. 1,670 ± 646 pg/108 cells TxB2-production).ConclusionsAspirin is a substrate for MRP4 and can be extruded from platelet through its transportation. Aspirin effect on COX-1 is little-related to MRP4-mediated aspirin transport in HV, but in CABG patients with MRP4 over-expression, its pharmacological inhibition enhances aspirin action in an efficient way.

In multicellular organisms, cell proliferation and death must be regulated to maintain tissue homeostasis. Many observations suggest that this regulation may be achieved, in part, by coupling the process of cell cycle progression and programmed cell death by using and controlling a shared set of factors. An argument in favor of a link between the cell cycle and apoptosis arises from the accumulated evidence that manipulation of the cell cycle may either prevent or induce an apoptotic response. This linkage has been recognized for tumor suppressor genes such as p53 and RB, the dominant oncogene, c-Myc, several cyclin-dependent kinases (Cdks) and their regulators. These proteins that function in proliferative pathways may also act to sensitize cells to apoptosis. Indeed, unregulated cell proliferation can result in pathologic conditions including neoplasias if it is not countered by the appropriate cell death. Translating the knowledge gained by studying the connection between cell death and cell proliferation may aid in identifying novel therapies to circumvent disease progression or improve clinical outcome.