Exendin-4 protects adipose-derived mesenchymal stem cells from apoptosis induced by hydrogen peroxide through the PI3K/Akt–Sfrp2 pathways

Adipose-derived mesenchymal stem cells (ADMSCs)-based therapy is a promising modality for the treatment of myocardial infarction in the future. However, the majority of transplanted cells are readily lost after transplantation because of hypoxia and oxidative stress. An efficient means to enhance the ability of ADMSCs to survive under pathologic conditions is required. In our study, we explored the effects of exendin-4 (Ex-4) on ADMSCs apoptosis in vitro induced by hydrogen peroxide, focusing in particular on mitochondrial apoptotic pathways and PI3K/Akt–secreted frizzled-related protein 2 (Sfrp2) survival signaling. We demonstrated that ADMSCs subjected to H₂O₂ for 12 h exhibited impaired mitochondrial function and higher apoptotic rate. However, Ex-4 (1–20 nM) preconditioning for 12 h could protect ADMSCs against H₂O₂-mediated apoptosis in a dose-dependent manner. Furthermore, Ex-4 pretreatment upregulated the levels of superoxide dismutase and glutathione as well as downregulating the production of reactive oxygen species and malondialdehyde. Western blots revealed that increased antiapoptotic proteins Bcl-2 and c-IAP1/2 as well as decreased proapoptotic proteins Bax and cytochrome c appeared in ADMSCs with Ex-4 incubation, which inhibited the caspase-9-involved mitochondrial apoptosis pathways with evidence showing inactivation of caspase-9/3 and preservation of mitochondrial membrane potential. Furthermore, we illustrated that Ex-4 enhanced Akt phosphorylation, which increased the expression of Sfrp2. Notably, blockade of the PI3K/Akt pathway or knockdown of Sfrp2 with siRNA obviously abolished the protective effects of Ex-4 on mitochondrial function and ADMSCs apoptosis under H₂O₂. In summary, this study confirmed that H₂O₂ induced ADMSCs apoptosis through mitochondria-dependent cell death pathways, and Ex-4 preconditioning may reduce such apoptosis of ADMSCs through the PI3K/Akt–Sfrp2 pathways.     

Induction of mitochondrial biogenesis protects against caspase-dependent and caspase-independent apoptosis in L6 myoblasts

Apoptotic signaling plays an important role in skeletal muscle degradation, atrophy, and dysfunction. Mitochondria are central executers of apoptosis by directly participating in caspase-dependent and caspase-independent cell death signaling. Given the important apoptotic role of mitochondria, altering mitochondrial content could influence apoptosis. Therefore, we examined the direct effect of modest, but physiological increases in mitochondrial biogenesis and content on skeletal muscle apoptosis using a cell culture approach. Treatment of L6 myoblasts with SNAP or AICAR (5 h/day for 5 days) significantly increased PGC-1, AIF, cytochrome c, and MnSOD protein content as well as MitoTracker staining. Following induction of mitochondrial biogenesis, L6 myoblasts displayed decreased sensitivity to apoptotic cell death as well as reduced caspase-3 and caspase-9 activation following exposure to staurosporine (STS) and C2-ceramide. L6 myoblasts with higher mitochondrial content also exhibited reduced apoptosis and AIF release following exposure to hydrogen peroxide (H₂O₂). Analysis of several key apoptosis regulatory proteins (ARC, Bax, Bcl-2, XIAP), antioxidant proteins (catalase, MnSOD, CuZnSOD), and reactive oxygen species (ROS) measures (DCF and MitoSOX fluorescence) revealed that these mechanisms were not responsible for the observed cellular protection. However, myoblasts with higher mitochondrial content were less sensitive to Ca^2 +-induced mitochondrial permeability transition pore formation (mPTP) and mitochondrial membrane depolarization. Collectively, these data demonstrate that increased mitochondrial content at physiological levels provides protection against apoptotic cell death by decreasing caspase-dependent and caspase-independent signaling through influencing mitochondrial Ca^2 +-mediated apoptotic events. Therefore, increasing mitochondrial biogenesis/content may represent a potential therapeutic approach in skeletal muscle disorders displaying increased apoptosis.     

Tyrosine phosphorylation of cytochrome c as a signaling event in frozen thawed buffalo spermatozoa at the cross-roads of capacitation and apoptosis

Considering the importance of cytochrome c in both life and death, it was of significant interest to investigate the expression of cytochrome c, its tyrosine phosphorylation status and immunolocalization patterns in a frozen-thawed buffalo sperm cell in comparison to in vitro capacitated [heparin (10 μg/ml) induced, for 4 h] and stress [apoptotic (10 μM staurosporine), oxidative (25 μM H₂O₂) and osmotic (180 mM NaCl) for 4 h] induced conditions. Proteins were subjected to immunoblotting and probed by using monoclonal anti-phosphotyrosine antibodies. A significant (p < 0.05) increase in expression of tyrosine phosphorylated cytochrome c was observed in capacitated buffalo sperm in comparison to frozen-thawed samples. cAMP protein kinase-A dependent and extracellular signal-regulated kinase independent tyrosine phosphorylation of cytochrome c was found during in vitro capacitation of buffalo spermatozoa. Localized increase in cytochrome c and tyrosine phosphorylated proteins were observed in frozen thawed and capacitated sperm. The information generated in this study can be used to understand the molecular mechanism of regulation of an apoptotic protein (cytochrome c) by tyrosine phosphorylation (a capacitation marker) in a frozen thawed sperm cell which could be a good target to combat apoptosis.     

Protective effects of purified safflower extract on myocardial ischemia in vivo and in vitro

Carthamus tinctorius L. (safflower) is one of the most commonly used Chinese herbal medicines to prevent and treat cardiac disease in clinical practice. However, the mechanisms responsible for such protective effects remain largely unknown. In this study, we investigated the anti-myocardial ischemia effects of a purified extract of C. tinctorius (ECT) both in vivo and in vitro. An animal model of myocardial ischemia injury was induced by left anterior descending coronary artery occlusion in adult rats. Pretreatment with ECT (100, 200, 400, 600 mg/kg body wt.) could protect the heart from ischemia injury by limiting infarct size and improving cardiac function. In the in vitro experiment, neonatal rat ventricular myocytes were incubated to test the direct cytoprotective effect of ECT against H₂O₂ exposure. Pretreatment with 100–400 μg/ml ECT prior to H₂O₂ exposure significantly increased cell viability as revealed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. ECT also markedly attenuated H₂O₂-induced cardiomyocyte apoptosis, as detected by Annexin V and PI double labeling with flow cytometry. The intracellular level of reactive oxygen species (ROS) was shown by 2′,7′-dichlorofluorescin diacetate (DCFH-DA), and ECT pretreatment significantly inhibited H₂O₂-induced ROS increase. We made a preliminary examination of the signaling cascade involved in ECT mediated anti-apoptotic effects. Phosphatidylinositol 3 kinase (PI3K) inhibitor (LY294002) blocked the cytoprotective effect conferred by ECT. Taken together, our findings provide the first evidence that the cardioprotective effects of ECT in myocardial ischemia operate partially through reducing oxidative stress induced damage and apoptosis. The protection is achieved by scavenging of ROS and mediating the PI3K signaling pathway.     

Mechanical stretch and prostaglandin E2 modulate critical signaling pathways in mouse podocytes

Elevated glomerular capillary pressure (Pgc) and hyperglycemia contribute to glomerular filtration barrier injury observed in diabetic nephropathy (DN). Previous studies showed that hypertensive conditions alone or in combination with a diabetic milieu impact podocyte cellular function which results in podocyte death, detachment or hypertrophy. The present study was aimed at uncovering the initial signaling profile activated by Pgc (mimicked by in vitro mechanical stretch), hyperglycemia (high glucose (HG), 25 mM d-glucose) and prostaglandin E₂ (PGE₂) in conditionally-immortalized mouse podocytes. PGE₂ significantly reduced the active form of AKT by selectively blunting its phosphorylation on S473, but not on T308. AKT inhibition by PGE₂ was reversed following either siRNA-mediated EP₄ knockdown, PKA inhibition (H89), or phosphatase inhibition (orthovanadate). Podocytes treated for 20 min with H₂O₂ (10^?4 M), which mimics reactive oxygen species generation by cells challenged by hyperglycemic or enhanced Pgc conditions, significantly increased the levels of active p38 MAPK, AKT, JNK and ERK1/2. Interestingly, stretch and PGE₂ each significantly reduced H₂O₂-mediated AKT phosphorylation and was reversed by pretreatment with orthovanadate while stretch alone reduced GSK-3β inhibitory phosphorylation at ser-9. Finally, mechanical stretch alone or in combination with HG, induced ERK1/2 and JNK activation, via the EGF receptor since AG1478, a specific EGF receptor kinase inhibitor, blocked this activation. These results show that cellular signaling in podocytes is significantly altered under diabetic conditions (i.e., hyperglycemia and increased Pgc). These changes in MAPKs and AKT activities might impact cellular integrity required for a functional glomerular filtration barrier thereby contributing to the onset of proteinuria in DN.     

β-Arrestin prevents cell apoptosis through pro-apoptotic ERK1/2 and p38 MAPKs and anti-apoptotic Akt pathways

Our previous studies have shown that β-arrestin 2 plays an anti-apoptotic effect. However, the mechanisms by which β-arrestin contribute to anti-apoptotic role remain unclear. In this study, we show that a deficiency of either β-arrestin 1 or β-arrestin 2 significantly increases serum deprivation (SD)-induced percentage of apoptotic cells. β-arrestin 2 deficient-induced apoptosis was inhibited by transfection with β-arrestin 2 full-length plasmid, revealing that SD-induced apoptosis is dependent on β-arrestin 2. Furthermore, in the absence of either β-arrestin 1 or β-arrestin 2 significantly enhances SD-induced the level of pro-apoptotic proteins, including cleaved caspase-3, extracellular-signal regulated kinase 1/2 (ERK1/2) and p38, members of mitogen-activated protein kinases (MAPKs). In addition, a deficiency of either β-arrestin 1 or β-arrestin 2 inhibits phosphorylation of Akt. The SD-induced changes in cleaved caspase-3, ERK1/2 and p38 MAPKs, Akt, and apoptotic cell numbers could be blocked by double knockout of β-arrestin 1/2. Our study thus demonstrates that β-arrestin inhibits cell apoptosis through pro-apoptotic ERK1/2 and p38 MAPKs and anti-apoptotic Akt signaling pathways.     

Cell death-resistance of differentiated myotubes is associated with enhanced anti-apoptotic mechanisms compared to myoblasts

Skeletal muscle atrophy is associated with elevated apoptosis while muscle differentiation results in apoptosis resistance, indicating that the role of apoptosis in skeletal muscle is multifaceted. The objective of this study was to investigate mechanisms underlying apoptosis susceptibility in proliferating myoblasts compared to differentiated myotubes and we hypothesized that cell death-resistance in differentiated myotubes is mediated by enhanced anti-apoptotic pathways. C₂C₁₂ myoblasts and myotubes were treated with H₂O₂ or staurosporine (Stsp) to induce cell death. H₂O₂ and Stsp induced DNA fragmentation in more than 50% of myoblasts, but in myotubes less than 10% of nuclei showed apoptotic changes. Mitochondrial membrane potential dissipation was detected with H₂O₂ and Stsp in myoblasts, while this response was greatly diminished in myotubes. Caspase-3 activity was 10-fold higher in myotubes compared to myoblasts, and Stsp caused a significant caspase-3 induction in both. However, exposure to H₂O₂ did not lead to caspase-3 activation in myoblasts, and only to a modest induction in myotubes. A similar response was observed for caspase-2, -8 and -9. Abundance of caspase-inhibitors (apoptosis repressor with caspase recruitment domain (ARC), and heat shock protein (HSP) 70 and -25 was significantly higher in myotubes compared to myoblasts, and in addition ARC was suppressed in response to Stsp in myotubes. Moreover, increased expression of HSPs in myoblasts attenuated cell death in response to H₂O₂ and Stsp. Protein abundance of the pro-apoptotic protein endonuclease G (EndoG) and apoptosis-inducing factor (AIF) was higher in myotubes compared to myoblasts. These results show that resistance to apoptosis in myotubes is increased despite high levels of pro-apoptotic signaling mechanisms, and we suggest that this protective effect is mediated by enhanced anti-caspase mechanisms.     

α7 nicotinic acetylcholine receptors control cytochrome c release from isolated mitochondria through kinase-mediated pathways

Nicotinic acetylcholine receptors are ligand-gated ion channels found in the plasma membrane of both excitable and non-excitable cells. Previously we reported that nicotinic receptors containing α7 subunits were present in the outer membranes of mitochondria to regulate the early apoptotic events like cytochrome c release. Here we show that signaling of mitochondrial α7 nicotinic receptors affects intramitochondrial protein kinases. Agonist of α7 nicotinic receptors PNU 282987 (30 nM) prevented the effect of phosphatidyl inositol-3-kinase inhibitor wortmannin, which stimulated cytochrome c release in isolated mouse liver mitochondria, and restored the Akt (Ser 473) phosphorylation state decreased by either 90 μM Ca²⁺ or wortmannin. The effect of PNU 282987 was similar to inhibition of calcium-calmodulin-dependent kinase II (upon 90 μM Ca²⁺) or of Src kinase(s) (upon 0.5 mM H₂O₂) and of protein kinase C. Cytochrome c release from mitochondria could be also attenuated by α7 nicotinic receptor antagonist methyllicaconitine or α7-specific antibodies. Allosteric modulator PNU 120526 (1 μM) did not improve the effect of agonist PNU 282987. Acetylcholine (1 μM) and methyllicaconitine (10 nM) inhibited superoxide release from mitochondria measured according to alkalization of Ca²⁺-containing medium. It is concluded that α7 nicotinic receptors regulate mitochondrial permeability transition pore formation through ion-independent mechanism involving activation of intramitochondrial PI₃K/Akt pathway and inhibition of calcium-calmodulin-dependent or Src-kinase-dependent signaling pathways.     

Evidence for a second messenger function of dUTP during Bax mediated apoptosis of yeast and mammalian cells

The identification of novel anti-apoptotic sequences has lead to new insights into the mechanisms involved in regulating different forms of programmed cell death. For example, the anti-apoptotic function of free radical scavenging proteins supports the pro-apoptotic function of Reactive Oxygen Species (ROS). Using yeast as a model of eukaryotic mitochondrial apoptosis, we show that a cDNA corresponding to the mitochondrial variant of the human DUT gene (DUT-M) encoding the deoxyuridine triphosphatase (dUTPase) enzyme can prevent apoptosis in yeast in response to internal (Bax expression) and to exogenous (H₂O₂ and cadmium) stresses. Of interest, cell death was not prevented under culture conditions modeling chronological aging, suggesting that DUT-M only protects dividing cells. The anti-apoptotic function of DUT-M was confirmed by demonstrating that an increase in dUTPase protein levels is sufficient to confer increased resistance to H₂O₂ in cultured C2C12 mouse skeletal myoblasts. Given that the function of dUTPase is to decrease the levels of dUTP, our results strongly support an emerging role for dUTP as a pro-apoptotic second messenger in the same vein as ROS and ceramide.     

Critical role of hydrogen peroxide signaling in the sequential activation of p38 MAPK and eNOS in laminar shear stress

Laminar shear stress (LSS) is a protective hemodynamic regulator of endothelial function and limits the development of atherosclerosis and other vascular wall diseases related to pathophysiological generation of reactive oxygen species. LSS activates several endothelial signaling responses, including the activation of MAPKs and eNOS. Here, we explored the mechanisms of activation of these key endothelial signaling pathways. Using the cone/plate model we found that LSS (12 dyn/cm²) rapidly promotes endothelial intracellular generation of superoxide and hydrogen peroxide (H₂O₂). Physiological concentrations of H₂O₂ (flux of 0.1 nM/min and 15 μM added extracellularly) significantly activated both eNOS and p38 MAPK. Pharmacological inhibition of NADPH oxidases (NOXs) and specific knockdown of NOX4 decreased LSS-induced p38 MAPK activation. Whereas the absence of eNOS did not alter LSS-induced p38 MAPK activation, pharmacological inhibition and knockdown of p38α MAPK blocked H₂O₂- and LSS-induced eNOS phosphorylation and reduced ^?NO levels. We propose a model in which LSS promotes the formation of signaling levels of H₂O₂, which in turn activate p38α MAPK and then stimulate eNOS, leading to increased ^?NO generation and protection of endothelial function.     

Ethyl 2-(benzylidene)-7-methyl-3-oxo-2,3-dihydro-5H-thiazolo[3,2-a]pyrimidine-6-carboxylate analogues as a new scaffold for protein kinase casein kinase 2 inhibitor

Protein kinase casein kinase 2 (PKCK2) is a constitutively active, growth factor-independent serine/threonine kinase, and changes in PKCK2 expression or its activity are reported in many cancer cells. To develop a novel PKCK2 inhibitor(s), we first performed cell-based phenotypic screening using 4000 chemicals purchased from ChemDiv chemical libraries (2000: randomly selected; 2000: kinase-biased) and performed in vitro kinase assay-based screening using hits found from the first screening. We identified compound 24 (C24)[(Z)-ethyl 5-(4-chlorophenyl)-2-(3,4-dihydroxybenzylidene)-7-methyl-3-oxo-3,5-dihydro-2H-thiazolo[3,2-a] pyrimidine-6-carboxylate] as a novel inhibitor of PKCK2 that is more potent and selective than 4,5,6,7-tetrabromobenzotriazole (TBB). In particular, compound 24 [half maximal inhibitory concentration (IC₅₀) = 0.56 μM] inhibited PKCK2 2.2-fold more efficiently than did TBB (IC₅₀ = 1.24 μM), which is quite specific toward PKCK2 with respect to ATP binding, in a panel of 31 human protein kinases. The K_i values of compound 24 and TBB for PKCK2 were 0.78 μM and 2.70 μM, respectively. Treatment of cells with compound 24 inhibited endogenous PKCK2 activity and showed anti-proliferative and pro-apoptotic effects against stomach and hepatocellular cancer cell lines more efficiently than did TBB. As expected, compound 24 also enabled tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-resistant cancer cells to be sensitive toward TRAIL. In comparing the molecular docking of compound 24 bound to PKCK2α versus previously reported complexes of PKCK2 with other inhibitors, our findings suggest a new scaffold for specific PKCK2α inhibitors. Thus, compound 24 appears to be a selective, cell-permeable, potent, and novel PKCK2 inhibitor worthy of further characterization.     

SIRT3 protects cardiomyocytes from oxidative stress-mediated cell death by activating NF-κB

Oxidative stress-mediated cell death in cardiomyocytes reportedly plays an important role in many cardiac pathologies. Our previous report demonstrated that mitochondrial SIRT3 plays an essential role in mediating cell survival in cardiac myocytes, and that resveratrol protects cardiomyocytes from oxidative stress-induced apoptosis by activating SIRT3. However, the exact mechanism by which SIRT3 prevents oxidative stress remains unknown. Here, we show that exposure of H9c2 cells to 50 μM H₂O₂ for 6 h caused a significant increase in cell death and the down-regulation of SIRT3. Reactive oxygen species (ROS)-mediated NF-κB activation was involved in this SIRT3 down-regulation. The SIRT3 activator, resveratrol, which is considered an important antioxidant, protected against H₂O₂-induced cell death, whereas the SIRT inhibitor, nicotinamide, enhanced cell death. Moreover, resveratrol negatively regulated H₂O₂-induced NF-κB activation, whereas nicotinamide enhanced H₂O₂-induced NF-κB activation. We also found that SOD2, Bcl-2 and Bax, the downstream genes of NF-κB, were involved in this pathological process. These results suggest that SIRT3 protects cardiomyocytes exposed to oxidative stress from apoptosis via a mechanism that may involve the NF-κB pathway.     

Overexpression of a 60-kDa heat shock protein enhances cytoprotective function of small intestinal epithelial cells

AimsWith the advancement of small intestinal (double balloon and capsule) endoscopy technology, incidence of small intestinal lesion caused by nonsteroidal anti-inflammatory drugs (NSAIDs) has been known to be high. However, therapy for small intestinal mucosal lesion has not yet been developed. Previous studies have shown that heat shock proteins (HSPs) are involved in cytoprotection mediated by their function as a molecular chaperone. In this study, we examined the effect of HSP60 or HSP70 overexpression on hydrogen peroxide-induced (H₂O₂) or indomethacin-induced cell damage in the small intestinal epithelial cells.Main methodscDNA of human HSP60 or HSP70 was transfected to rat small intestinal (IEC-6) cells, and HSP60- or HSP70-overexpressing cells were cloned. IEC-6 cells transfected with vector only were used as control cells. These cells were treated with H₂O₂ (0–0.14 mM) or indomethacin (0–2.5 mM). The cell viability was determined by MTT-assay. Cell necrosis was evaluated by LDH-release assay. Further, apoptosis was evaluated by caspases-3/7 activity and TUNEL assay.Key findingsCell viability after H₂O₂ or indomethacin treatment was significantly higher in HSP60-overexpressing cells compared with that in control cells and HSP60-overexpressing cells. Apoptotic cells were also reduced in HSP60-overexpressing. Conclusion: These results indicate that HSP60 plays an important role in protecting small intestinal mucosal cells from H₂O₂-induced or indomethacin-induced cell injury. HSP70-overexpressing cells did not show anti-apoptotic ability.SignificanceThese findings possibly suggest that function of each HSP is different in the small intestine. Therefore, for the therapy of small intestinal mucosal lesion, HSP60-induction therapy could be a new therapeutic strategy.     

Enhancement of PPAR-β activity by repetitive low-grade H2O2 stress protects human umbilical vein endothelial cells from subsequent oxidative stress-induced apoptosis

Repetitive stress has been shown to up-regulate antioxidant defense and increase survival after subsequent oxidative injury. The up-regulation of antioxidant defense has been identified as an underlying cause of the apoptosis-inhibitory effects exerted by repetitive stress. However, it remains unclear what the important signaling mechanisms are by which cells preexposed to low-grade stress deal with apoptosis-inducing stress. In this study, we repetitively stressed human umbilical vein endothelial cells (HUVECs) through multiple exposures to a low dose (30 μM) of H₂O₂ in culture for 4 weeks. We then examined the effects of repetitive stress on PPAR-β expression and activity as well as the role of PPAR-β in the protective potency of repetitive stress. Our results show that repetitive stress enhances PPAR-β expression and activity, thereby inhibiting oxidative stress-induced apoptosis. Further, PPAR-β-directed antisense oligonucleotides reduced the PPAR-β protein content, enhanced the H₂O₂-mediated apoptosis, and ablated the protective effect of repetitive low-grade H₂O₂ stress. The specific PPAR-β agonist L-165041 significantly potentiated the apoptosis induced by H₂O₂ (p < 0.05) and increased the protective effect of repetitive stress. These findings indicate that repetitive low-grade H₂O₂ stress protects HUVECs from subsequent oxidative stress-induced apoptosis by enhancing PPAR-β expression and activity.     

Signal transduction pathways through cytoprotective, apoptotic and hypertrophic stimuli: a comparative study in adult cardiac myocytes

In response to pathophysiological stresses, cardiac myocytes undergo hypertrophic growth or apoptosis. Multiple signalling pathways have been implicated in these responses and among them, kinases such as mitogen‐activated protein kinases (MAPKs) and Akt. However, the distinction between signalling pathways originally believed to be specific for either hypertrophy, apoptosis or cell survival is fading. The existing data, coming from different experimental systems, often are conflicting. In this study, we sought to compare aspects of intracellular signalling activated by diverse stimuli in a single experimental system, adult rat cardiac myocytes. Furthermore, we assessed the role of these stimuli in eliciting a particular cell phenotype, i.e. whether they promote hypertrophy, cell survival or apoptosis. The results demonstrate that the hypertrophic agonist phenylephrine is the most potent activator of MAPKs/mitogen and stress‐ activated kinase MSK1, although its effect on Akt phosphorylation is relatively minor. The pro‐apoptotic concentration of H₂O₂ activates strongly both MAPKs and PI3K/Akt pathways. Insulin‐like growth factor‐1 has a minimal effect on phosphorylation of MAPKs/MSK1, but it is a potent activator of Akt. In conclusion, hypertrophic, pro‐survival or apoptotic stimuli operate through the same signalling pathways with different time course and amplitude of kinase activation. Thus, to determine the effect of different stimuli on cell fate, it is important to assess signalling pathways as a network and not as a single pathway. Copyright © 2011 John Wiley & Sons, Ltd.     

Rapamycin can restore the negative regulatory function of transforming growth factor beta 1 in high grade lymphomas

TGF-β1 (transforming growth factor beta 1) is a negative regulator of lymphocytes, inhibiting proliferation and switching on the apoptotic program in normal lymphoid cells. Lymphoma cells often lose their sensitivity to proapoptotic/anti-proliferative regulators such as TGF-β1. Rapamycin can influence both mTOR (mammalian target of rapamycin) and TGF-β signaling, and through these pathways it is able to enhance TGF-β induced anti-proliferative and apoptotic responses. In the present work we investigated the effect of rapamycin and TGF-β1 combination on cell growth and on TGF-β and mTOR signalling events in lymphoma cells.Rapamycin, an inhibitor of mTORC1 (mTOR complex 1) did not elicit apoptosis in lymphoma cells; however, the combination of rapamycin with exogenous TGF-β1 induced apoptosis and restored TGF-β1 dependent apoptotic machinery in several lymphoma cell lines with reduced TGF-β sensitivity in vitro. In parallel, the phosphorylation of p70 ribosomal S6 kinase (p70S6K) and ribosomal S6 protein, targets of mTORC1, was completely eliminated. Knockdown of Smad signalling by Smad4 siRNA had no influence on apoptosis induced by the rapamycin + TGF-β1, suggesting that this effect is independent of Smad signalling. However, apoptosis induction was dependent on early protein phosphatase 2A (PP2A) activity, and in part on caspases. Rapamycin + TGF-β1 induced apoptosis was not completely eliminated by a caspase inhibitor.These results suggest that high mTOR activity contributes to TGF-β resistance and lowering mTORC1 kinase activity may provide a tool in high grade B-cell lymphoma therapy by restoring the sensitivity to normally available regulators such as TGF-β1.     

Proteomic-based identification of Apg-2 as a therapeutic target for chronic myeloid leukemia

The oncogenic BCR/ABL tyrosine kinase induces constitutive enhanced “spontaneous” DNA damage and unfaithful repair in Philadelphia chromosome positive leukemia cells. Here, we investigated the changes of protein profile in H₂O₂-induced DNA damage/repair in BaF3-MIGR1 and BaF3-BCR/ABL cells through a proteomic strategy consisting of two-dimensional gel electrophoresis (2-DE) coupled with MALDI-TOF mass spectrometry. In total, 41 spots were differentially expressed and 13 proteins were identified with further MS analysis. Two essential proteins, Proto-oncogene tyrosine–protein kinase ABL1 (c-ABL) and Heat shock 70 kDa protein 4 (Apg-2), were confirmed by Western blot and showed consistent changes with proteomic results. Moreover, functional analysis demonstrated that inhibition of Apg-2 not only decreased cell proliferation, but also induced cell apoptosis in BCR/ABL positive cells (BaF3-BCR/ABL, BaF3-BCR/ABL^T315I). We also proved that Apg-2 inhibition aggravated H₂O₂ induced damage in BCR/ABL positive cells, and enhanced the sensitivity of BaF3-BCR/ABL^T315I to STI571. Taken together, the findings in this work provide us with some clues to a better understanding of the molecular mechanisms underlying BCR/ABL in the DNA damage/repair processes and demonstrated that Apg-2 would be a valid target for anti-leukemia drug development.