Phosphorylated protein phosphatase 2A determines poor outcome in patients with metastatic colorectal cancer.

BACKGROUND: Protein phosphatase 2A (PP2A) is a tumour suppressor frequently inactivated in human cancer and its tyrosine-307 phosphorylation has been reported as a molecular inhibitory mechanism. METHODS: Expression of phosphorylated PP2A (p-PP2A) was evaluated in 250 metastatic colorectal cancer (CRC) patients. Chi-square, Kaplan-Meier and Cox analyses were used to determine correlations with clinical and molecular parameters and impact on clinical outcomes. RESULTS: High p-PP2A levels were found in 17.2% cases and were associated with ECOG performance status (P=0.001) and presence of synchronous metastasis at diagnosis (P=0.035). This subgroup showed substantially worse overall survival (OS) (median OS, 6.0 vs 26.2 months, P<0.001) and progression-free survival (PFS) (median PFS, 3.8 vs 13.3 months, P<0.001). The prognostic impact of p-PP2A was particularly evident in patients aged <70 years (P<0.001). Multivariate analysis revealed that p-PP2A retained its prognostic impact for OS (hazard ratio 2.7; 95% confidence interval, 1.8-4.1; P<0.001) and PFS (hazard ratio 3.0; 95% confidence interval, 1.8-5.0; P<0.001). CONCLUSIONS: Phosphorylated PP2A is an alteration that determines poor outcome in metastatic CRC and represents a novel potential therapeutic target in this disease, thus enabling to define a subgroup of patients who could benefit from future treatments based on PP2A activators.

Influence of exercise on the circulating levels and macrophage production of IL-1beta and IFNgamma affected by metabolic syndrome: an obese Zucker rat experimental animal model.

Regular physical activity is recognized as a non-pharmacological treatment of genetic obesity and type-II diabetes, and based on the "anti-inflammatory" effects of exercise, it has been also proposed for improving the "chronic low-grade inflammation" in metabolic syndrome (MS). The aim of the present work was to evaluate the effects of an habitual exercise program (running, 5 days/week for 35 min at 35 cm/s for 14 weeks) and of a bout of acute exercise (running, for 35 min at 35 cm/s) on MS-associated disorders in the pro-inflammatory cytokines IL-1beta and IFNgamma. The study was carried out on obese Zucker rats (fa/fa). The obese rats presented higher circulating concentrations and constitutive macrophage production (in the absence of antigenic stimulus) of IL-1beta (but not of IFNgamma). But their production of both IL-1beta and IFNgamma by lipopolysaccharide (LPS)-stimulated macrophages was lower than that of the control lean rats. Our protocol of exercise training did not modify the circulating concentration and constitutive macrophage release of either IL-1beta or IFNgamma in the obese rats, but increased the production of both cytokines by LPS-stimulated macrophages. The single bout of acute exercise only increased the release of IL-1beta by the LPS-stimulated macrophages from obese rats, in both sedentary and trained animals. The results indicated that: (1) circulating levels and constitutive production of IL-1beta by macrophages are deregulated in rats with MS, and (2) IL-1beta and IFNgamma production by macrophages in response to antigenic stimulus (LPS) is impaired in the obese animals, and this MS-associated disorder is improved by the program of habitual exercise training.

Responses of rat myocardial antioxidant defences and heat shock protein HSP72 induced by 12 and 24-week treadmill training.

AIM: The purpose of this study was to determine the effects of both a short (12 weeks) and a long-term (24 weeks) endurance treadmill-training programme on the levels of oxidative stress markers, the activity of the enzymatic antioxidants, and the content of the 72 kDa heat shock protein (HSP72) in rat myocardium. METHODS: Thirty male Wistar rats were randomly assigned to exercise trained (n = 16) and sedentary (n = 14) groups. After 12 week of training, eight rats were killed while the remaining eight continued the training programme until 24 week. RESULTS: Seven sedentary controls were killed together with each trained group. Levels of thiobarbituric acid-reactive substances (TBARS), protein carbonyls, and total and oxidized glutathione (tGSH and GSSG) in myocardial homogenates were unchanged by training irrespective of the protocol duration. However, an increased content of the oxidative stress biomarkers was detected in hearts from both the 24-week trained rats and their sedentary controls when compared with their corresponding 12-week groups. The antioxidant enzymatic activities total and mitochondrial superoxide dismutase (tSOD and mtSOD, respectively), glutathione peroxidase (GPX) and glutathione reductase (GR), remained unchanged after the 12-week training period whereas a significant increase in tSOD and mtSOD activities (18%, P < 0.05) was observed in heart homogenates of 24-week trained animals as compared with their sedentary controls. HSP72 expression levels were not significantly modified after 12 week of training but a threefold increase was detected after 24 week (P < 0.05). CONCLUSION: These results demonstrate that a long-term endurance training (24 weeks) induced discrete increases in antioxidant enzyme activities in rat myocardium and elicited a marked enhancement in HSP72 expression levels. However, a shorter training programme (12 weeks), was not effective in increasing heart antioxidant defences.

Protein variants of skeletal muscle regulatory myosin light chain isoforms: prevalence in mammals, generation and transitions during muscle remodelling.

The regulatory myosin light chains (rMLCs) of mammalian skeletal muscle display protein diversity arising from the existence of different isotypes and protein phosphorylation. Two-dimensional electrophoresis and immunoblotting allowed us to identify three variants of the slow and fast rMLC isoforms (designated LC2s, LC2s1, LC2s2, and LC2f, LC2f1, LC2f2, respectively, from less to more acidic). This study aimed to characterize their prevalence among different species and muscle types, the mechanism(s) of their generation and their transitions during fast-to-slow fibre type switching. In vitro dephosphorylation and back-phosphorylation experiments and mass spectrometric analysis of tryptic digests indicate that: (1) both acidic variants, within each isoform, contain a phosphorylated peptide, (2) all variants of each isoform share identical tryptic peptides, (3) only one phosphopeptide is present per isoform, and (4) the intermediate-acidic variants of both isoforms contain the same peptide in their phosphorylated and non-phosphorylated forms. The data indicate that the triad pattern of variants results from two partially superimposed doublets of phosphorylated/non-phosphorylated pairs. Continuous, low-frequency electrical stimulation of rat extensor digitorum longus muscle changed the relative proportions of variants within each isoform towards those of the soleus. It is suggested that the doublets of phosphorylated/non-phosphorylated pairs are involved in rMLC exchange during sarcomere remodelling.

Anabolic steroid and gender-dependent modulation of cytosolic HSP70s in fast- and slow-twitch skeletal muscle.

Besides their clinical uses, anabolic steroids (AASs) are self-administered by athletes to improve muscle mass and sports performance. The biological basis for their presumed effectiveness at suprapharmacological doses, however, remains uncertain. Since the expression of high levels of some stress proteins (HSPs) has been associated with an increased tolerance to stress and chronic exercise up-regulates HSP72 in skeletal muscle, this investigation was aimed at testing whether the administration of suprapharmacological doses of AASs, either alone or in conjunction with chronic exercise, induced changes in HSP72. Nandrolone decanoate (ND), an estrene derivative, but not stanozolol (ST), a derivative of the androstane series, up-regulated the levels of HSP72 and changed the proportions of various charge variants of the cytosolic HSP70s in sedentary and exercise-trained rats, exclusively in fast-twitch fibres. Since the expression of HSP73-levels in skeletal muscle was dependent on gender but not on muscle type, and that of HSP72-levels was muscle type specific but gender-independent, ND effects on cytosolic HSP70s could not be explained solely by a functional relationship with sex steroids. The reported results indicate that, by up-regulating the expression levels of HSP72 in fast-twitch fibres, nandrolone decanoate could contribute to improving the tolerance of skeletal muscle to high-intensity training.

Stress proteins of 70 kDa in chronically exercised skeletal muscle.

Skeletal muscle fibres of untrained animals experience a stress response following exercise. This study was aimed at investigating whether chronic exercise modulates stress proteins of 70 kDa (HSP70s) in skeletal muscle. In the soleus muscle of Wistar rats, adherence to an incremental programme of treadmill running (IPTR) of 3 months duration up-regulated the levels of the beta-subunit of the mitochondrial F1-ATPase and those of HSP72, GRP75 and GRP78. Neither beta-F1-ATPase nor sarcoplasmic reticulum Ca2+-ATPase levels changed with training in the extensor digitorum longus (EDL) muscle. However, HSP70s increased during training. In soleus muscle slices of animals sacrificed 3 days after completing the IPTR, HSP72 and GRP75 were synthesized at lower rates than in sedentary animals while the GRP78 synthesis rate increased. Trained, rested animals also experienced a stress response following acute exercise of lower intensity than that of the actual training sessions. The data suggest that up-regulation of HSP70s by chronic exercise depends upon continued physical activity. Furthermore, the inverse correlation between levels and rates of synthesis of HSP72 during rest periods suggests the operation of a feedback regulatory loop aimed at reestablishing the threshold levels characteristic of unstressed fibres.

Muscle fibre stress in response to exercise: synthesis, accumulation and isoform transitions of 70-kDa heat-shock proteins.

Heat-shock or stress proteins (HSPs) are considered to play an essential role in protecting cells from stress and preparing them to survive new environmental challenges. This study investigates the induction kinetics of synthesis and accumulation of 70-kDa stress proteins in the soleus and extensor digitorum longus (EDL) muscles of the rat following exercise, as well as the isoform transitions that take place during the post-exercise period. Relative synthesis rates (referred to constitutively expressed stress protein HSP73) of the 70-kDa heat-shock proteins were greatly enhanced after a single bout of exercise in both muscles. They peaked early in the post-exercise period and returned to resting levels after approximately 5-6 h. The levels of the inducible stress protein HSP72 in the EDL rose only transiently following exercise, while its accumulation in the soleus was more continuous and stable. The amount of HSP73 increased only transiently in both muscle types after exercise. The constitutive expression of the stress protein HSP72 in the soleus muscle was much higher than in the EDL and other tissues, while that of HSP73 was relatively constant among tissues. Rat skeletal muscle HSP72 and HSP73 were made up of at least three isoforms of the same molecular mass and very close isoelectric points, although only one radiolabelled isoform was detected. The relative proportion of the most abundant isoforms of HSP72, isoforms 1 and 2, as well as their ratio (isoform 2/isoform 1), increased during the post-exercise period. Since isoform 2 of HSP72 partially disappeared after incubating soleus muscle extracts of exercised rats with alkaline phosphatase, these data indicate that phosphorylation of HSP72 is an early event in the stress response of skeletal muscle to exercise stress.

Anabolic steroids and lymphocyte function in sedentary and exercise-trained rats.

The effects of the administration of suprapharmacological doses of anabolic steroids (AASs) on the immune system were examined in sedentary and exercise-trained rats by testing mobility and proliferative response in cultures of thymus and spleen-derived lymphocytes. Male Wistar rats were exercise-trained following two programmes of treadmill running of 3 months duration, differing in intensity, in the absence of treatment or with simultaneous i.m. administration of a suprapharmacological dose (10 mg/kg/week) of nandrolone decanoate (ND) or stanozolol (ST) during the past two months. At this dose ND reduced body weight gain, promoted a redistribution of immune cells from thymus to spleen, impaired lymphocyte mobility and inhibited the mitogen-induced proliferative response (about 90% inhibition for thymus-derived cells). Stanozolol (ST) treatment was without effect on body weight gain, but it also induced a redistribution of lymphocytes and modified the in vitro lymphocyte activity, although less severely than ND. Application of the high-intensity training programme reduced lymphocyte mobility and proliferation in vitro and a simultaneous treatment with anabolic steroids further impaired some of the immune cell responses. Application of the endurance-directed training programme, however, did not reduce mobility or mitogen-induced proliferation of lymphocytes, and normalized the activity of these cells in anabolic steroid-treated rats. So, endurance exercise, contrary to high-intensity training, could counteract the apparent negative effects of high doses of androgens on lymphocyte function.

Effect of clenbuterol on the modulation of noradrenaline release in the rat tail artery.

1. Exposure of rat tail arteries to clenbuterol, a beta 2-adrenoceptor agonist, for 20 or 90 min, did not change or increase, respectively, tritium overflow induced by electrical field stimulation in arteries preincubated with [3H]-noradrenaline (NA). This facilitatory effect was antagonized by propranolol. 2. Phentolamine increased the evoked overflow four-fold, which was not modified by 90 min incubation with clenbuterol. In rats pretreated with clenbuterol for 2 weeks, the stimulated overflow was not enhanced by this beta 2-agonist, and the increase produced by phentolamine was markedly diminished. 3. Contractile responses induced by electrical field stimulation were not modified or increased (only at low frequencies) by preincubation with clenbuterol for 20 or 90 min, respectively. This effect was inhibited by propranolol. 4. In arteries precontracted with 5-hydroxytryptamine, clenbuterol (10 nM-10 microM) produced small relaxations, which were reduced by propranolol plus phentolamine and not modified by phentolamine or 90 min exposure to clenbuterol. 5. These results indicate that prolonged exposure of rat tail arteries to clenbuterol produces a facilitation of NA release mediated by activation of presynaptic beta 2-adrenoceptors, which may be involved on the enhancement of contractile responses to electrical stimulation induced by clenbuterol. However, chronic treatment with this beta-agonist desensitizes these receptors.