Gabriella Marfe

Efficient production by sperm-mediated gene transfer of human decay accelerating factor (hDAF) transgenic pigs for xenotransplantation

A large number of hDAF transgenic pigs to be used for xenotransplantation research were generated by using sperm-mediated gene transfer (SMGT). The efficiency of transgenesis obtained with SMGT was much greater than with any other method. In the experiments reported, up to 80% of pigs had the transgene integrated into the genome. Most of the pigs carrying the hDAF gene transcribed it in a stable manner (64%). The great majority of pigs that transcribed the gene expressed the protein (83%). The hDAF gene was transmitted to progeny. Expression was stable and found in caveolae as it is in human cells. The expressed gene was functional based on in vitro experiments performed on peripheral blood mononuclear cells. These results show that our SMGT approach to transgenesis provides an efficient procedure for studies involving large animal models.

Kaempferol induces apoptosis in two different cell lines via Akt inactivation, Bax and SIRT3 activation, and mitochondrial dysfunction†

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 µM 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. J. Cell. Biochem. 106: 643–650, 2009.

Hypoxia-increased RAGE and P2X7R expression regulates tumor cell invasion through phosphorylation of Erk1/2 and Akt and nuclear translocation of NF-κB

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.

Sphingosine Kinase 1 Overexpression Contributes to Cetuximab Resistance in Human Colorectal Cancer Models

Purpose: Although the anti–EGF receptor (EGFR) monoclonal antibody cetuximab is an effective strategy in colorectal cancer therapy, its clinical use is limited by intrinsic or acquired resistance. Alterations in the “sphingolipid rheostat”—the balance between the proapoptotic molecule ceramide and the mitogenic factor sphingosine-1-phosphate (S1P)—due to sphingosine kinase 1 (SphK1) overactivation have been involved in resistance to anticancer-targeted agents. Moreover, cross-talks between SphK1 and EGFR-dependent signaling pathways have been described. Experimental design: We investigated SphK1 contribution to cetuximab resistance in colorectal cancer, in preclinical in vitro/in vivo models, and in tumor specimens from patients. Results: SphK1 was found overexpressed and overactivated in colorectal cancer cells with intrinsic or acquired resistance to cetuximab. SphK1 contribution to resistance was supported by the demonstration that SphK1 inhibition by N,N-dimethyl-sphingosine or silencing via siRNA in resistant cells restores sensitivity to cetuximab, whereas exogenous SphK1 overexpression in sensitive cells confers resistance to these agents. Moreover, treatment of resistant cells with fingolimod (FTY720), a S1P receptor (S1PR) antagonist, resulted in resensitization to cetuximab both in vitro and in vivo, with inhibition of tumor growth, interference with signal transduction, induction of cancer cells apoptosis, and prolongation of mice survival. Finally, a correlation between SphK1 expression and cetuximab response was found in colorectal cancer patients. Clin Cancer Res; 19(1); 138–47. ©2012 AACR.

SIRT3 protects from hypoxia and staurosporine-mediated cell death by maintaining mitochondrial membrane potential and intracellular pH

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 (ΔΨmt), 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 ΔΨmt, intracellular pH and mitochondrial-regulated apoptotic pathways.

The effect of marathon on mRNA expression of anti-apoptotic and pro-apoptotic proteins and sirtuins family in male recreational long-distance runners

BackgroundA 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.).ResultsBlood 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).ConclusionThese data suggest that the physiological load imposed in amateur runners during marathon attenuates the extent of apoptosis and may interfere with sirtuin expression.

SIRT1 silencing confers neuroprotection through IGF-1 pathway activation

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.

A new clinical approach: Use of blood-derived stem cells (BDSCs) for superficial digital flexor tendon injuries in horses

AIMS In this study, we present an innovative therapy using stem cells that were obtained from the peripheral blood of racehorses affected by uninduced superficial digital flexor tendon (SDFT) injuries. MAIN METHODS Blood-derived stem cells (BDSCs) were generated from the blood samples of three horses in the presence of macrophage colony-stimulating factor (M-CSF). The racehorses received a single autologous BDSC treatment, which resulted in the successful repair of the tendons injuries. KEY FINDINGS The results demonstrated that the BDSCs injection into the damaged tendon stimulated the regeneration of normal tissue. Furthermore, a relationship may exist between the speed and the quality of new tissue formation and the welfare and management of the treated animals. SIGNIFICANCE This study demonstrates that stem cell technology offers new tools for tissue repair that in many cases is considered incurable, and provides additional evidence that BDScs injections increase the speed and quality of the regeneration process in different animal tissues.

Sphingosine kinase 1 overexpression is regulated by signaling through PI3K, AKT2, and mTOR in imatinib-resistant chronic myeloid leukemia cells

OBJECTIVE As a better understanding of the molecular basis of carcinogenesis has emerged, oncogene-specific cell-signaling pathways have been successfully targeted to treat human malignances. Despite impressive advances in oncogene-directed therapeutics, genetic instability in cancer cells often manifest acquired resistance. This is particularly noted in the use of tyrosine kinase inhibitors therapies and not more evident than for chronic myeloid leukemia. Therefore, it is of great importance to understand the molecular mechanisms affecting cancer cell sensitivity and resistance to tyrosine kinase inhibitors. MATERIALS AND METHODS In this study, we used continuous exposure to stepwise increasing concentrations of imatinib (0.6-1 μM) to select imatinib-resistant K562 cells. RESULTS Expression of BCR-ABL increased both at RNA and protein levels in imatinib-resistant cell lines. Furthermore, expression levels of sphingosine kinase 1 (SphK1) were increased significantly in resistant cells, channeling sphingoid bases to the SphK1 pathway and activating sphingosine-1-phosphate-dependent tyrosine phosphorylation pathways that include the adaptor protein Crk. The partial inhibition of SphK1 activity by N,N-dimethylsphingosine or expression by small interfering RNA increased sensitivity to imatinib-induced apoptosis in resistant cells and returned BCR-ABL to baseline levels. To determine the resistance mechanism-induced SphK1 upregulation, we used pharmacological inhibitors of the phosphoinositide 3-kinase/AKT/mammalian target of rapamycin signaling pathway and observed robust downmodulation of SphK1 expression and activity when AKT2, but not AKT1 or AKT3, was suppressed. CONCLUSIONS These results demonstrate that SphK1 is upregulated in imatinib-resistant K562 cells by a pathway contingent on a phosphoinositide 3-kinase/AKT2/mammalian target of rapamycin signaling pathway. We propose that SphK1 plays an important role in development of acquired resistance to imatinib in chronic myeloid leukemia cell lines.

Efficiency of transgenesis using sperm-mediated gene transfer: generation of hDAF transgenic pigs.

SINCE the beginning of this century, replacement of failing human organs with their animal counterparts has been an interesting topic of debate for writers and scientists. In the 1960s, prolonged survival after kidney transplantation from chimpanzee to human was obtained in the United States and Europe. Nevertheless, both the progressive improvement in surgical technique and in immunosuppressant therapy and the availability of cadaveric organs and living donation have reduced the interest in xenotransplantation. Because of the increasing requests for organs and the lack of donors to meet that need, xenotransplantation has become a reliable option again for temporary organ replacement (eg, of heart or liver) before definitive transplant. However, primates such as chimpanzees and baboons are expensive, can carry important zoonoses, and their use is burdened by ethical implications. A better choice for xenotransplantation might be offered by swine, which are closer to humans for anatomic and metabolic features. Discordant transplantation is associated with humoral hyperacute rejection, due to preformed antibodies and complement system activation (both classical and alternative pathway). This results in massive and irreversible vascular damage and cellular necrosis. Expression of the species-specific complement activation inhibitors could prevent this kind of rejection. Organs harvested from pigs transgenic for human decay accelerating factor (hDAF or CD55), membrane cofactor protein (MCP or CD46), and CD59 could be more efficiently grafted into human recipients. Therefore, a number of research teams, including our group, have generated pigs transgenic for human negative regulators of the complement cascade. This research has been aimed to generate hDAF transgenic swine with high efficiency and reproducibility. This goal has been reached through the innovative method of sperm-mediated gene transfer (SMGT), which was developed about 10 years ago by our group. Data presented in this paper show that hDAF-positive individuals can also be used as founders of stable lines of F1 generation transgenic pigs. MATERIALS AND METHODS hDAF Transgenic F1 Offspring Generation