Dose escalation to high-risk sub-volumes based on non-invasive imaging of hypoxia and glycolytic activity in canine solid tumors:a feasibility study

INTRODUCTION: Glycolytic activity and hypoxia are associated with poor prognosis and radiation resistance. Including both the tumor uptake of 2-deoxy-2-[(18) F]-fluorodeoxyglucose (FDG) and the proposed hypoxia tracer copper(II)diacetyl-bis(N(4))-methylsemithio-carbazone (Cu-ATSM) in targeted therapy planning may therefore lead to improved tumor control. In this study we analyzed the overlap between sub-volumes of FDG and hypoxia assessed by the uptake of (64)Cu-ATSM in canine solid tumors, and evaluated the possibilities for dose redistribution within the gross tumor volume (GTV). MATERIALS AND METHODS: Positron emission tomography/computed tomography (PET/CT) scans of five spontaneous canine solid tumors were included. FDG-PET/CT was obtained at day 1, (64)Cu-ATSM at day 2 and 3 (3 and 24 h pi.). GTV was delineated and CT images were co-registered. Sub-volumes for 3 h and 24 h (64)Cu-ATSM (Cu3 and Cu24) were defined by a threshold based method. FDG sub-volumes were delineated at 40% (FDG40) and 50% (FDG50) of SUV(max). The size of sub-volumes, intersection and biological target volume (BTV) were measured in a treatment planning software. By varying the average dose prescription to the tumor from 66 to 85 Gy, the possible dose boost (D( B )) was calculated for the three scenarios that the optimal target for the boost was one, the union or the intersection of the FDG and (64)Cu-ATSM sub-volumes. RESULTS: The potential boost volumes represented a fairly large fraction of the total GTV: Cu3 49.8% (26.8-72.5%), Cu24 28.1% (2.4-54.3%), FDG40 45.2% (10.1-75.2%), and FDG50 32.5% (2.6-68.1%). A BTV including the union (∪) of Cu3 and FDG would involve boosting to a larger fraction of the GTV, in the case of Cu3∪FDG40 63.5% (51.8-83.8) and Cu3∪FDG50 48.1% (43.7-80.8). The union allowed only a very limited D( B ) whereas the intersection allowed a substantial dose escalation. CONCLUSIONS: FDG and (64)Cu-ATSM sub-volumes were only partly overlapping, suggesting that the tracers offer complementing information on tumor physiology. Targeting the combined PET positive volume (BTV) for dose escalation within the GTV results in a limited D( B ). This suggests a more refined dose redistribution based on a weighted combination of the PET tracers in order to obtain an improved tumor control

Acute hyperglycemia abolishes cardioprotection by remote ischemic perconditioning

BACKGROUND: Remote ischemic perconditioning (RIPerC) has a promising therapeutic insight to improve the prognosis of acute myocardial infarction. Chronic comorbidities such as diabetes are known to interfere with conditioning interventions by modulating cardioprotective signaling pathways, such as e.g., mTOR pathway and autophagy. However, the effect of acute hyperglycemia on RIPerC has not been studied so far. Therefore, here we investigated the effect of acute hyperglycemia on cardioprotection by RIPerC. METHODS: Wistar rats were divided into normoglycemic (NG) and acute hyperglycemic (AHG) groups. Acute hyperglycemia was induced by glucose infusion to maintain a serum glucose concentration of 15-20 mM throughout the experimental protocol. NG rats received mannitol infusion of an equal osmolarity. Both groups were subdivided into an ischemic (Isch) and a RIPerC group. Each group underwent reversible occlusion of the left anterior descending coronary artery (LAD) for 40 min in the presence or absence of acute hyperglycemia. After the 10-min LAD occlusion, RIPerC was induced by 3 cycles of 5-min unilateral femoral artery and vein occlusion and 5-min reperfusion. After 120 min of reperfusion, infarct size was measured by triphenyltetrazolium chloride staining. To study underlying signaling mechanisms, hearts were harvested for immunoblotting after 35 min in both the NG and AHG groups. RESULTS: Infarct size was significantly reduced by RIPerC in NG, but not in the AHG group (NG + Isch: 46.27 +/- 5.31 % vs. NG + RIPerC: 24.65 +/- 7.45 %, p < 0.05; AHG + Isch: 54.19 +/- 4.07 % vs. 52.76 +/- 3.80 %). Acute hyperglycemia per se did not influence infarct size, but significantly increased the incidence and duration of arrhythmias. Acute hyperglycemia activated mechanistic target of rapamycine (mTOR) pathway, as it significantly increased the phosphorylation of mTOR and S6 proteins and the phosphorylation of AKT. In spite of a decreased LC3II/LC3I ratio, other markers of autophagy, such as ATG7, ULK1 phopsphorylation, Beclin 1 and SQSTM1/p62, were not modulated by acute hyperglycemia. Furthermore, acute hyperglycemia significantly elevated nitrative stress in the heart (0.87 +/- 0.01 vs. 0.50 +/- 0.04 microg 3-nitrotyrosine/mg protein, p < 0.05). CONCLUSIONS: This is the first demonstration that acute hypreglycemia deteriorates cardioprotection by RIPerC. The mechanism of this phenomenon may involve an acute hyperglycemia-induced increase in nitrative stress and activation of the mTOR pathway

Receptor-Specific Mechanisms Regulate Phosphorylation of AKT at Ser473: Role of RICTOR in β1 Integrin-Mediated Cell Survival

A tight control over AKT/PKB activation is essential for cells, and they realise this in part by regulating the phosphorylation of Ser473 in the “hydrophobic motif” of the AKT carboxy-terminal region. The RICTOR-mTOR complex (TORC2) is a major kinase for AKT Ser473 phosphorylation after stimulation by several growth factors, in a reaction proposed to require p21-activated kinase (PAK) as a scaffold. However, other kinases may catalyse this reaction in stimuli-specific manners. Here we characterised the requirement of RICTOR, ILK, and PAK for AKT Ser473 phosphorylation downstream of selected family members of integrins, G protein-coupled receptors, and tyrosine-kinase receptors and analysed the importance of this phosphorylation site for adhesion-mediated survival. siRNA-mediated knockdown in HeLa and MCF7 cells showed that RICTOR-mTOR was required for phosphorylation of AKT Ser473, and for efficient phosphorylation of the downstream AKT targets FOXO1 Thr24 and BAD Ser136, in response to β1 integrin-stimulation. ILK and PAK1/2 were dispensable for these reactions. RICTOR knockdown increased the number of apoptotic MCF7 cells on β1 integrin ligands up to 2-fold after 24 h in serum-free conditions. β1 integrin-stimulation induced phosphorylation of both AKT1 and AKT2 but markedly preferred AKT2. RICTOR-mTOR was required also for LPA-induced AKT Ser473 phosphorylation in MCF7 cells, but, interestingly, not in HeLa cells. PAK was needed for the AKT Ser473 phosphorylation in response to LPA and PDGF, but not to EGF. These results demonstrate that different receptors utilise different enzyme complexes to phosphorylate AKT at Ser473, and that AKT Ser473 phosphorylation significantly contributes to β1 integrin-mediated anchorage-dependent survival of cells

A Glutathione Peroxidase, Intracellular Peptidases and the TOR Complexes Regulate Peptide Transporter PEPT-1 in C. elegans

The intestinal peptide transporter PEPT-1 in Caenorhabditis elegans is a rheogenic H+-dependent carrier responsible for the absorption of di- and tripeptides. Transporter-deficient pept-1(lg601) worms are characterized by impairments in growth, development and reproduction and develop a severe obesity like phenotype. The transport function of PEPT-1 as well as the influx of free fatty acids was shown to be dependent on the membrane potential and on the intracellular pH homeostasis, both of which are regulated by the sodium-proton exchanger NHX-2. Since many membrane proteins commonly function as complexes, there could be proteins that possibly modulate PEPT-1 expression and function. A systematic RNAi screening of 162 genes that are exclusively expressed in the intestine combined with a functional transport assay revealed four genes with homologues existing in mammals as predicted PEPT-1 modulators. While silencing of a glutathione peroxidase surprisingly caused an increase in PEPT-1 transport function, silencing of the ER to Golgi cargo transport protein and of two cytosolic peptidases reduced PEPT-1 transport activity and this even corresponded with lower PEPT-1 protein levels. These modifications of PEPT-1 function by gene silencing of homologous genes were also found to be conserved in the human epithelial cell line Caco-2/TC7 cells. Peptidase inhibition, amino acid supplementation and RNAi silencing of targets of rapamycin (TOR) components in C. elegans supports evidence that intracellular peptide hydrolysis and amino acid concentration are a part of a sensing system that controls PEPT-1 expression and function and that involves the TOR complexes TORC1 and TORC2

Mycobacterium marinum antagonistically induces an autophagic response while repressing the autophagic flux in a TORC1- and ESX-1-dependent manner.

Autophagy is a eukaryotic catabolic process also participating in cell-autonomous defence. Infected host cells generate double-membrane autophagosomes that mature in autolysosomes to engulf, kill and digest cytoplasmic pathogens. However, several bacteria subvert autophagy and benefit from its machinery and functions. Monitoring infection stages by genetics, pharmacology and microscopy, we demonstrate that the ESX-1 secretion system of Mycobacterium marinum, a close relative to M. tuberculosis, upregulates the transcription of autophagy genes, and stimulates autophagosome formation and recruitment to the mycobacteria-containing vacuole (MCV) in the host model organism Dictyostelium. Antagonistically, ESX-1 is also essential to block the autophagic flux and deplete the MCV of proteolytic activity. Activators of the TORC1 complex localize to the MCV in an ESX-1-dependent manner, suggesting an important role in the manipulation of autophagy by mycobacteria. Our findings suggest that the infection by M. marinum activates an autophagic response that is simultaneously repressed and exploited by the bacterium to support its survival inside the MCV

Emerging evidence of a link between the polycystins and the mTOR pathways

Autosomal dominant polycystic kidney disease (ADPKD) is a genetic disease characterized by the formation of renal cysts. This disease can be caused by mutations in two genes, PKD1 and PKD2, which encode polycystin-1 (PC-1) and -2 (PC-2), respectively