Nutrition, Nitrogen Requirements, Exercise and Chemotherapy-Induced Toxicity in Cancer Patients. A puzzle of Contrasting Truths?

Amino acids can modulate cell metabolism and control cell fate by regulating cell survival and cell death. The molecular mechanisms involved are mediated by the mTOR complexes mTORC1 and mTORC2 activity. These complexes are finely regulated and the continuous advancement of the knowledge on their composition and function is revealing that their balance may represent the condition that determines the cell fate. This is important for normal healthy cells but it is becoming clear, and it is even more important, that the balance of the mTORCs activity may also condition the cell fate of cancer cells. Here, we discuss the evidences supporting the amino acids supplementation as a cancer fighting weapon and a possible strategy to counteract the myocyte toxicity associated with chemotherapy, possibly by tipping the balance of mTORCs activity.

Effect of antihypertensive treatments on insulin signalling in lympho-monocytes of essential hypertensive patients: a pilot study.

It was previously demonstrated that metabolic syndrome in humans is associated with an impairment of insulin signalling in circulating mononuclear cells. At least in animal models of hypertension, angiotensin-converting enzyme (ACE) inhibitors and angiotensin receptor blockers (ARB) may correct alterations of insulin signalling in the skeletal muscle. In the first study, we investigated the effects of a 3-month treatment with an ARB with additional PPARγ agonist activity, telmisartan, or with a dihydropyridine calcium channel blocker, nifedipine, on insulin signalling in patients with mild-moderate essential hypertension. Insulin signalling was evaluated in mononuclear cells by isolating them through Ficoll-Paque density gradient centrifugation and protein analysis by Western Blot. An increased expression of mTOR and of phosphorylated (active) mTOR (p-mTOR) was observed in patients treated with telmisartan, but not in those treated with nifedipine, while both treatments increased the cellular expression of glucose transporter type 4 (GLUT-4). We also investigated the effects of antihypertensive treatment with two drug combinations on insulin signalling and oxidative stress. Twenty essential hypertensive patients were included in the study and treated for 4 weeks with lercanidipine. Then they were treated for 6 months with lercanidipine + enalapril or lercanidipine + hydrochlorothiazide. An increased expression of insulin receptor, GLUT-4 and an increased activation of p70S6K1 were observed during treatment with lercanidipine + enalapril but not with lercanidipine + hydrochlorothiazide. In conclusion, telmisartan and nifedipine are both effective in improving insulin signalling in human hypertension; however, telmisartan seems to have broader effects. The combination treatment lercanidipine + enalapril seems to be more effective than lercanidipine + hydrochlorothiazide in activating insulin signalling in human lympho-monocytes.

Mechanisms inducing low bone density in Duchenne muscular dystrophy in mice and humans.

Patients affected by Duchenne muscular dystrophy (DMD) and dystrophic MDX mice were investigated in this study for their bone phenotype and systemic regulators of bone turnover. Micro-computed tomographic (µCT) and histomorphometric analyses showed reduced bone mass and higher osteoclast and bone resorption parameters in MDX mice compared with wild-type mice, whereas osteoblast parameters and mineral apposition rate were lower. In a panel of circulating pro-osteoclastogenic cytokines evaluated in the MDX sera, interleukin 6 (IL-6) was increased compared with wild-type mice. Likewise, DMD patients showed low bone mineral density (BMD) Z-scores and high bone-resorption marker and serum IL-6. Human primary osteoblasts from healthy donors incubated with 10% sera from DMD patients showed decreased nodule mineralization. Many osteogenic genes were downregulated in these cultures, including osterix and osteocalcin, by a mechanism blunted by an IL-6-neutralizing antibody. In contrast, the mRNAs of osteoclastogenic cytokines IL6, IL11, inhibin-ßA, and TGFß2 were increased, although only IL-6 was found to be high in the circulation. Consistently, enhancement of osteoclastogenesis was noted in cultures of circulating mononuclear precursors from DMD patients or from healthy donors cultured in the presence of DMD sera or IL-6. Circulating IL-6 also played a dominant role in osteoclast formation because ex vivo wild-type calvarial bones cultured with 10% sera of MDX mice showed increase osteoclast and bone-resorption parameters that were dampen by treatment with an IL-6 antibody. These results point to IL-6 as an important mediator of bone loss in DMD and suggest that targeted anti-IL-6 therapy may have a positive impact on the bone phenotype in these patients.

The glycosaminoglycan-binding domain of PRELP acts as a cell type-specific NF-kappaB inhibitor that impairs osteoclastogenesis.

Proline/arginine-rich end leucine-rich repeat protein (PRELP) is a glycosaminoglycan (GAG)- and collagen-binding anchor protein highly expressed in cartilage, basement membranes, and developing bone. We observed that PRELP inhibited in vitro and in vivo mouse osteoclastogenesis through its GAG-binding domain ((hbd)PRELP), involving (a) cell internalization through a chondroitin sulfate- and annexin II-dependent mechanism, (b) nuclear translocation, (c) interaction with p65 nuclear factor kappaB (NF-kappaB) and inhibition of its DNA binding, and (d) impairment of NF-kappaB transcriptional activity and reduction of osteoclast-specific gene expression. (hbd)PRELP does not disrupt the mitogen-activated protein kinase signaling nor does it impair cell survival. (hbd)PRELP activity is cell type specific, given that it is internalized by the RAW264.7 osteoclast-like cell line but fails to affect calvarial osteoblasts, bone marrow macrophages, and epithelial cell lines. In vivo, (hbd)PRELP reduces osteoclast number and activity in ovariectomized mice, underlying its physiological and/or pathological importance in skeletal remodeling.

Global transcriptome analysis in mouse calvarial osteoblasts highlights sets of genes regulated by modeled microgravity and identifies a "mechanoresponsive osteoblast gene signature".

Mechanical unloading is known to be detrimental for the skeleton, but the underlying molecular mechanisms are not fully elucidated. We performed global transcriptome analysis of mouse calvarial osteoblasts grown for 5 days at unit gravity (1g) or under modeled microgravity (0.008g) in the NASA-developed rotating wall vessel (RWV) bioreactor. Elaboration of gene profiling data evidenced that, among the >20,000 gene probes evaluated, 45 genes were significantly up-regulated (cut-off >2) and 88 were down-regulated (cut-off <0.5) in modeled microgravity versus 1g. This set of regulated genes includes genes involved in osteoblast differentiation, function, and osteoblast-osteoclast cross-talk, as well as new genes not previously correlated with bone metabolism. Microarray data were validated for subsets of genes by real-time RT-PCR, Western blot, or functional analysis. The significantly modulated genes were then clustered using the GOTM (Gene Ontology Tree Machine) software. This analysis evidenced up-regulation of genes involved in the induction of apoptosis, in response to stress and in the activity of selected growth factors. Other molecular functions, such as extracellular matrix structural constituent, glycosaminoglycan/heparin-binding activity, and other growth factor activity, were instead down-regulated. We finally matched our transcriptome results with other public global gene profiles obtained in loading and unloading conditions, identifying 10 shared regulated genes which could represent an "osteoblast mechanoresponsive gene signature."

Beta-Arrestin2 regulates RANKL and ephrins gene expression in response to bone remodeling in mice.

PTH-stimulated intracellular signaling is regulated by the cytoplasmic adaptor molecule beta-arrestin. We reported that the response of cancellous bone to intermittent PTH is reduced in beta-arrestin2(-/-) mice and suggested that beta-arrestins could influence the bone mineral balance by controlling RANKL and osteoprotegerin (OPG) gene expression. Here, we study the role of beta-arrestin2 on the in vitro development and activity of bone marrow (BM) osteoclasts (OCs) and Ephrins ligand (Efn), and receptor (Eph) mRNA levels in bone in response to PTH and the changes of bone microarchitecture in wildtype (WT) and beta-arrestin2(-/-) mice in models of bone remodeling: a low calcium diet (LoCa) and ovariectomy (OVX). The number of PTH-stimulated OCs was higher in BM cultures from beta-arrestin2(-/-) compared with WT, because of a higher RANKL/OPG mRNA and protein ratio, without directly influencing osteoclast activity. In vivo, high PTH levels induced by LoCa led to greater changes in TRACP5b levels in beta-arrestin2(-/-) compared with WT. LoCa caused a loss of BMD and bone microarchitecture, which was most prominent in beta-arrestin2(-/-). PTH downregulated Efn and Eph genes in beta-arrestin2(-/-), but not WT. After OVX, vertebral trabecular bone volume fraction and trabecular number were lower in beta-arrestin2(-/-) compared with WT. Histomorphometry showed that OC number was higher in OVX-beta-arrestin2(-/-) compared with WT. These results indicate that beta-arrestin2 inhibits osteoclastogenesis in vitro, which resulted in decreased bone resorption in vivo by regulating RANKL/OPG production and ephrins mRNAs. As such, beta-arrestins should be considered an important mechanism for the control of bone remodeling in response to PTH and estrogen deprivation.

Modeled microgravity stimulates osteoclastogenesis and bone resorption by increasing osteoblast RANKL/OPG ratio.

Mechanical unloading causes detrimental effects on the skeleton, but the underlying mechanisms are still unclear. We investigated the effect of microgravity on osteoblast ability to regulate osteoclastogenesis. Mouse osteoblast primary cultures were grown for 24 h at unit gravity or under simulated microgravity, using the NASA-developed Rotating Wall Vessel bioreactor. Conditioned media (CM) from osteoblasts subjected to microgravity increased osteoclastogenesis and bone resorption in mouse bone marrow cultures. In these osteoblasts, the RANKL/OPG ratio was higher relative to 1g. Consistently, treatment with high concentrations of OPG-inhibited osteoclastogenesis and bone resorption in the presence of CM arising from osteoblasts cultured under microgravity. Microgravity failed to affect osteoblast differentiation and function in the time frame of the experiment, as we found no effect on alkaline phosphatase mRNA and activity, nor on Runx2, osteocalcin, osteopontin, and collagen1A2 mRNA expression. In contrast, microgravity induced a time dependent increase of ERK-1/2 phosphorylation, while phospho-p38 and phospho-JNK remained unchanged. Apoptosis, revealed by bis-benzimide staining, was similar among the various gravity conditions, while it was increased under microgravity after treatment with the MEK-1/2 inhibitor, PD98059, suggesting a protection role by ERK-1/2 against cell death. In conclusion, microgravity is capable to indirectly stimulate osteoclast formation and activity by regulating osteoblast secretion of crucial regulatory factors such as RANKL and OPG. We hypothesize that this mechanism could contribute to bone loss in individuals subjected to weightlessness and other unloading conditions.

Temporin L: antimicrobial, haemolytic and cytotoxic activities, and effects on membrane permeabilization in lipid vesicles.

The temporins are a family of small, linear antibiotic peptides with intriguing biological properties. We investigated the antibacterial, haemolytic and cytotoxic activities of temporin L (FVQWFSKFLGRIL-NH2), isolated from the skin of the European red frog Rana temporaria. The peptide displayed the highest activity of temporins studied to date, against both human erythrocytes and bacterial and fungal strains. At variance with other known temporins, which are mainly active against Gram-positive bacteria, temporin L was also active against Gram-negative strains such as Pseudomonas aeruginosa A.T.C.C. 15692 and Escherichia coli D21 at concentrations comparable with those that are microbiocidal to Gram-positive bacteria. In addition, temporin L was cytotoxic to three different human tumour cell lines (Hut-78, K-562 and U-937), causing a necrosis-like cell death, although sensitivity to the peptide varied markedly with the specific cell line tested. A study of the interaction of temporin L with liposomes of different lipid compositions revealed that the peptide causes perturbation of bilayer integrity of both neutral and negatively charged membranes, as revealed by the release of a vesicle-encapsulated fluorescent marker, and that the action of the peptide is modulated to some extent by membrane lipid composition. In particular, the presence of negatively charged lipids in the model bilayer inhibits the lytic power of temporin L. We also show that the release of fluorescent markers caused by temporin L is size-dependent and that the peptide does not have a detergent-like effect on the membrane, suggesting that perturbation of bilayer organization takes place on a local scale, i.e. through the formation of pore-like openings.