Laura Terraneo

Antitumour activity of melatonin in a mouse model of human prostate cancer: relationship with hypoxia signalling.

Melatonin is known to exert antitumour activity in several types of human cancers, but the underlying mechanisms as well as the efficacy of different doses of melatonin are not well defined. Here, we test the hypothesis whether melatonin in the nanomolar range is effective in exerting antitumour activity in vivo and examine the correlation with the hypoxia signalling mechanism, which may be a major molecular mechanism by which melatonin antagonizes cancer. To test this hypothesis, LNCaP human prostate cancer cells were xenografted into seven‐wk‐old Foxn1nu/nu male mice that were treated with melatonin (18 i.p. injections of 1 mg/kg in 41 days). Saline‐treated mice served as control. We found that the melatonin levels in plasma and xenografted tissue were 4× and 60× higher, respectively, than in control samples. Melatonin tended to restore the redox imbalance by increasing expression of Nrf2. As part of the phenotypic response to these perturbations, xenograft microvessel density was less in melatonin‐treated animals, indicative of lower angiogenesis, and the xenograft growth rate was slower (P < 0.0001). These changes were accompanied by a reduced expression of Ki67, elevated expression of HIF‐1α and increased phosphorylation of Akt in melatonin than saline‐treated mice. We conclude that the beneficial effect of melatonin in reducing cancer growth in vivo was evident at melatonin plasma levels as low as 4 nm and was associated with decreased angiogenesis. Higher HIF‐1α expression in xenograft tissue indicates that the antitumour effect cannot be due to a postulated antihypoxic effect, but may stem from lower angiogenesis potential.

Protein modulation in mouse heart under acute and chronic hypoxia

Exploring cellular mechanisms underlying beneficial and detrimental responses to hypoxia represents the object of the present study. Signaling molecules controlling adaptation to hypoxia (HIF‐1α), energy balance (AMPK), mitochondrial biogenesis (PGC‐1α), autophagic/apoptotic processes regulation and proteomic dysregulation were assessed. Responses to acute hypoxia (AH) and chronic hypoxia (CH) in mouse heart proteome were detected by 2‐D DIGE, mass spectrometry and antigen–antibody reactions. Both in AH and CH, the results indicated a deregulation of proteins related to sarcomere stabilization and muscle contraction. Neither in AH nor in CH the HIF‐1α stabilization was observed. In AH, the metabolic adaptation to lack of oxygen was controlled by AMPK activation and sustained by an up‐regulation of adenosylhomocysteinase and acetyl‐CoA synthetase. AH was characterized by the mitophagic protein Bnip 3 increment. PGC‐1α, a master regulator of mitochondrial biogenesis, was down‐regulated. CH was characterized by the up‐regulation of enzymes involved in antioxidant defense, in aldehyde bio‐product detoxification and in misfolded protein degradation. In addition, a general down‐regulation of enzymes controlling anaerobic metabolism was observed. After 10 days of hypoxia, cardioprotective molecules were substantially decreased whereas pro‐apoptotic molecules increased accompained by down‐regulation of specific target proteins.

Chronic systemic hypoxia promotes LNCaP prostate cancer growth in vivo

Solid tumors contain underperfused regions where hypoxia‐inducible factor‐1α (HIF‐1α) over‐expression induces hypoxia adaptation and cell proliferation. We test the hypothesis that systemic hypoxia promotes prostate cancer growth in vivo and examine HIF‐1α centrality in this effect.

Expression of carbohydrate-antigen sialyl-Lewis a on colon cancer cells promotes xenograft growth and angiogenesis in nude mice.

We investigated the role of carbohydrate antigen sialyl-Lewis a (sLea), an E-selectin ligand and epitope of tumor marker CA19.9, in the development of xenografts in nude mice. To this end, animals were inoculated with the human colon cancer cell line HCT-15, expressing no Lewis antigens, or with a clone expressing sLea (HCT-15-T5). The size of HCT-15-T5 xenografts appeared larger than those of HCT-15 and their average weight was over twice bigger. In both xenografts the mitotic index was found elevated, as determined by Ki-67 assay, and no apoptosis was detected in the tumor cells by both caspase 8 or TUNEL assays. Some apoptotic signals were instead detected in the vessels. Conversely, microvessel density, determined through CD-31 immunohistochemistry, was found 3.2-folds bigger in HCT-15-T5 xenografts (p<0.012). Only the membranes of HCT-15-T5 cells grown as xenografts reacted intensively with the anti CA19.9 antibody 1116-NS-19-9 by immunofluorescence, but not by immunohistochemistry. Unknown structures were instead stained by such technique in both xenografts, as were in mouse tissues not expressing the antigen and in human colon adenocarcinoma. We conclude that expression of sLea on the surface of colon cancer cells improves xenograft growth and is associated with enhanced angiogenesis, while immunohistochemistry with 1116-NS-19-9 antibody appears not suitable to determine CA19.9 expression.

Impact of the Phosphatidylinositide 3-Kinase Signaling Pathway on the Cardioprotection Induced by Intermittent Hypoxia

Background Exposure to intermittent hypoxia (IH) may enhance cardiac function and protects heart against ischemia-reperfusion (I/R) injury. To elucidate the underlying mechanisms, we developed a cardioprotective IH model that was characterized at hemodynamic, biochemical and molecular levels. Methods Mice were exposed to 4 daily IH cycles (each composed of 2-min at 6-8% O2 followed by 3-min reoxygenation for 5 times) for 14 days, with normoxic mice as controls. Mice were then anesthetized and subdivided in various subgroups for analysis of contractility (pressure-volume loop), morphology, biochemistry or resistance to I/R (30-min occlusion of the left anterior descending coronary artery (LAD) followed by reperfusion and measurement of the area at risk and infarct size). In some mice, the phosphatidylinositide 3-kinase (PI3K) inhibitor wortmannin was administered (24 µg/kg ip) 15 min before LAD. Results We found that IH did not induce myocardial hypertrophy; rather both contractility and cardiac function improved with greater number of capillaries per unit volume and greater expression of VEGF-R2, but not of VEGF. Besides increasing the phosphorylation of protein kinase B (Akt) and the endothelial isoform of NO synthase with respect to control, IH reduced the infarct size and post-LAD proteins carbonylation, index of oxidative damage. Administration of wortmannin reduced the level of Akt phosphorylation and worsened the infarct size. Conclusion We conclude that the PI3K/Akt pathway is crucial for IH-induced cardioprotection and may represent a viable target to reduce myocardial I/R injury.

Impact of hemoglobin concentration and affinity for oxygen on tissue oxygenation: the case of hemoglobin-based oxygen carriers.

In patients undergoing exchange-transfusion with hemoglobin (Hb)-based oxygen (O₂) carriers (HBOC), native Hb coexists with newly transfused Hb. The two Hb types share the same arterial and venous PO₂, but their affinities for O₂ vary. A simple spreadsheet model is described aiming at evaluating the contribution of each Hb type to the overall O₂ transport characteristics as a function of the batch Hb concentration and O₂ affinity in the HBOC solution, of the fraction of exchange-transfused blood/HBOC, and of the arterial PO₂. This model helps to yield a quantitative estimate of how tissues with high or low O₂ extraction respond to the changes cited above. The results show that the higher the exchange-transfusion ratio, the O₂ transport to tissues becomes progressively impaired. However, this effect is more critical at low batch Hb concentration and high O₂ affinity of the HBOC, especially for tissues/organs with high O₂ extraction, whereas the arterial PO₂ does not appear as critical.

Autophagy in Normal and Abnormal Early Human Pregnancies

Autophagy is an inducible catabolic process by which cells degrade and recycle materials to survive stress, starvation, and hypoxia. The aim of this study was to evaluate autophagy at the fetal–maternal interface, to assess autophagy involvement during the early phase of human gestation, and to explore autophagic modification in case of early abnormal pregnancy outcome. Specimens were collected from first-trimester normal gestations undergoing legal termination of pregnancy and first-trimester sporadic spontaneous miscarriages. Autophagy was studied in villous and decidual samples by transmission electron microscopy, immunohistochemistry, immunofluorescence, and Western blotting. Autophagy markers were found in cytotrophoblast, syncytiotrophoblast, extravillous trophoblast, and decidual stromal cells. Autophagy is physiologically involved in early normal gestation. Compared with normal pregnancy, spontaneous miscarriage presents an increase in autophagy expression in villous specimens due to an increment in concentration of autophagic vacuole in syncytiotrophoblast, suggesting a cytoprotective mechanism of the cells to respond to microenvironmental challenge.

Impact of acellular hemoglobin-based oxygen carriers on brain apoptosis in rats

Extracellular hemoglobin (Hb)‐based oxygen carriers (HBOCs) are under extensive consideration as oxygen therapeutics. Their effects on cellular mechanisms related to apoptosis are of particular interest, because the onset of proapoptotic pathways may give rise to tissue damage.

Brain adaptation to hypoxia and hyperoxia in mice

Aims Hyperoxic breathing might lead to redox imbalance and signaling changes that affect cerebral function. Paradoxically, hypoxic breathing is also believed to cause oxidative stress. Our aim is to dissect the cerebral tissue responses to altered O2 fractions in breathed air by assessing the redox imbalance and the recruitment of the hypoxia signaling pathways. Results Mice were exposed to mild hypoxia (10%O2), normoxia (21%O2) or mild hyperoxia (30%O2) for 28 days, sacrificed and brain tissue excised and analyzed. Although one might expect linear responses to %O2, only few of the examined variables exhibited this pattern, including neuroprotective phospho- protein kinase B and the erythropoietin receptor. The major reactive oxygen species (ROS) source in brain, NADPH oxidase subunit 4 increased in hypoxia but not in hyperoxia, whereas neither affected nuclear factor (erythroid-derived 2)-like 2, a transcription factor that regulates the expression of antioxidant proteins. As a result of the delicate equilibrium between ROS generation and antioxidant defense, neuron apoptosis and cerebral tissue hydroperoxides increased in both 10%O2 and 30%O2, as compared with 21%O2. Remarkably, the expression level of hypoxia-inducible factor (HIF)−2α (but not HIF-1α) was higher in both 10%O2 and 30%O2 with respect to 21%O2 Innovation Comparing the in vivo effects driven by mild hypoxia with those driven by mild hyperoxia helps addressing whether clinically relevant situations of O2 excess and scarcity are toxic for the organism. Conclusion Prolonged mild hyperoxia leads to persistent cerebral damage, comparable to that inferred by prolonged mild hypoxia. The underlying mechanism appears related to a model whereby the imbalance between ROS generation and anti-ROS defense is similar, but occurs at higher levels in hypoxia than in hyperoxia.

In vivo hyperoxia induces hypoxia-inducible factor-1α overexpression in LNCaP tumors without affecting the tumor growth rate

Hypoxia is a recognized cause for solid tumors malignancy and resistance, probably via hypoxia-induced overexpression of the hypoxia-inducible factor (HIF)-1α, major modulator of the cell response to oxygen deprivation. Although hyperoxia, the opposite condition, may represent a key issue to assess this paradigm, its effect on tumor growth and HIF-1α expression remains unclear. To test whether hyperoxia and hypoxia have divergent effects, and to better focus into the role of HIF-1α in vivo, athymic mice xenografted with LNCaP cells were exposed for 28 days to atmospheres containing 10, 21 or 30% O2. Whereas the xenografts grew twice faster in hypoxia, their growth rates in hyperoxia and normoxia were similar. To analyze the involved molecular mechanisms, we performed various assays in xenograft tissues. Faster xenografts growth in hypoxia was associated with higher phosphorylation of protein kinase B (Akt) and higher expression of Ki67, both related with pro-survival and cell proliferation pathways. By contrast, the expression level of HIF-1α was similar in normoxia and hypoxia, but paradoxically twice higher in hyperoxia. The protein level of the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) was also higher in hyperoxia, suggesting marked cell response to redox imbalance. Whereas both the vascular-endothelial growth factor (VEGF) and its receptor VEGF-R2 were overexpressed in hyperoxia, the tissue hemoglobin content was not increased, despite a slight reduction in vascularization. As a whole, this data indicates that the xenografts growth rate was independent of HIF-1α expression level, suggesting that in an in vivo setting alternative more effective proliferative paths associated with the cell response to the redox imbalance may override the paths linked to HIF-1α signaling.