Levels Of Phosphorylated p70S6K Research Articles

PI3K inhibition sensitizes EGFR wild‑type NSCLC cell lines to erlotinib chemotherapy

Tyrosine kinase inhibitors (TKIs) bring significant benefits for patients with cancers harboring epidermal growth factor receptor (EGFR) mutations. However, after treatment for a certain period, most patients ultimately acquire resistance. Numerous studies indicated that PI3K has an important role in tumor cell growth and drug sensitivity. Furthermore, inhibition of PI3K may lead to sensitization of non-small cell lung cancer (NSCLC) cells to EGFR-TKIs. The aim of the present study was to explore whether LY294002, an inhibitor of PI3K, is able to improve the sensitivity of NSCLC cell lines with wild-type EGFR to the EGFR-TKI erlotinib. An MTT assay was used to examine the effect of combined treatment with LY294002 and erlotinib on cell survival of two EGFR wild-type NSCLC cell lines, NCI-H661 and NCI-H460. Furthermore, flow cytometry was used to assess apoptosis in NCI-H661 and NCI-H460 cells after treatment with erlotinib and LY294002. In addition, the expression of downstream proteins was detected by western blot analysis. The results indicated that the number of viable NCI-H661 and NCI-H460 cells was dose-dependently reduced by erlotinib or LY294002. Compared to treatment with erlotinib alone, the cell apoptosis was enhanced if combined treatment of erlotinib and LY294002 was performed in NCI-H661 cells. Furthermore, combination treatment of erlotinib and LY294002 resulted in a significant reduction of phosphorylated p70S6K levels in NCI-H661 [PI3K catalytic subunit alpha (PI3KCA) wild-type] cells. However, this phenomenon was not observed in the NCI-H460 cell line (PIK3CA mutant-type). In conclusion, the present study indicated that inhibition of PI3K may have the potential to improve the sensitivity of NSCLC cells to an EGFR-TKI. However, the therapeutic effect may depend on the mutation status of PIK3CA.

Metformin counteracts the effects of FSH on rat Sertoli cell proliferation.

Metformin (MET) is one of the most widely used anti-hyperglycemic agents for treating patients with type 2 diabetes and it has started to be used in pediatric population at ages when Sertoli cells are still proliferating. It is well known that follicle-stimulating hormone (FSH) is the major Sertoli cell mitogen. The aim of the study is to investigate a possible effect of MET, which has been shown to have anti-proliferative properties, on FSH regulation of postnatal Sertoli cell proliferation and on the molecular mechanisms involved in this regulation. The present study was performed in eight-day-old rat Sertoli cell cultures. The results obtained show that MET in the presence of FSH increases phosphorylated acetyl-CoA carboxylase and decreases phosphorylated p70S6K levels. Moreover, we show that MET decreases FSH-stimulated Sertoli cell proliferation, and this decrease is accompanied by a reduction in FSH-stimulated Ccnd1 and Ccnd2 expression and an increase in cell cycle inhibitor p21Cip expression. Altogether, these results suggest that MET can, at least in part, counteract the effect of FSH on postnatal Sertoli cell proliferation.

Fingolimod Exerts only Temporary Antiepileptogenic Effects but Longer-Lasting Positive Effects on Behavior in the WAG/Rij Rat Absence Epilepsy Model.

One of the major challenges in the epilepsy field is identifying disease-modifying drugs in order to prevent or delay spontaneous recurrent seizure onset or to cure already established epilepsy. It has been recently reported that fingolimod, currently approved for the treatment of relapsing-remitting multiple sclerosis, has demonstrated antiepileptogenic effects in 2 different preclinical models of acquired epilepsy. However, to date, no data exist regarding the role of fingolimod against genetic epilepsy. Therefore, we have addressed this issue by studying the effects of fingolimod in Wistar Albino Glaxo/Rijswijk (WAG/Rij) rats, a well-established genetic model of absence epilepsy, epileptogenesis, and neuropsychiatric comorbidity. Our results have demonstrated that an early long-term treatment with fingolimod (1mg/kg/day), started before absence seizure onset, has both antiepileptogenic and antidepressant-like effects in WAG/Rij rats. However, these effects were transitory, as 5months after treatment discontinuation, both absence seizure and depressive like-behavior returned to control levels. Furthermore, a temporary reduction of mTOR signaling pathway activity, indicated by reduced phosphorylated mammalian target of rapamycin and phosphorylated p70S6k levels, and by increased phosphorylated Akt in WAG/Rij rats of 6months of age accompanied the transitory antiepileptogenic effects of fingolimod. Surprisingly, fingolimod has demonstrated longer-lasting positive effects on cognitive decline in this strain. This effect was accompanied by an increased acetylation of lysine 8 of histone H4 (at both 6 and 10months of age). In conclusion, our results support the antiepileptogenic effects of fingolimod. However, the antiepileptogenic effects were transitory. Moreover, fingolimod might also have a positive impact on animal behavior and particularly in protecting the development of memory decline.

Differential roles of the infralimbic and prelimbic areas of the prefrontal cortex in reconsolidation of a traumatic memory

Studies about reconsolidation of conditioned fear memories have shown that pharmacological manipulation at memory reactivation can attenuate or enhance the subsequent expression of the conditioned fear response.Here we examined the effects of a single injection of the mTOR inhibitor rapamycin (Rap) into the infralimbic (IL) and prelimbic (PL) areas [which compose the ventromedial prefrontal cortex (PFC)] on reconsolidation and extinction of a traumatic fear memory.We found opposite effects of Rap infused into the PL and IL on reconsolidation and extinction: intra-PL Rap and systemic Rap impaired reconsolidation and facilitated extinction whereas intra-IL Rap enhanced reconsolidation and impaired extinction. These effects persisted at least 10 days after reactivation. Shock exposure induced anxiety-like behavior and impaired working memory and intra-IL and –PL Rap normalized these effects. Finally, when measured after fear retrieval, shocked rats exhibited reduced and increased phosphorylated p70s6K levels in the IL and basolateral amygdala, respectively. No effect on phosphorylated p70s6K levels was observed in the PL.The study points to the differential roles of the IL and PL in memory reconsolidation and extinction. Moreover, inhibiting mTOR via rapamycin following reactivation of a fear memory may be a novel approach in attenuating enhanced fear memories.

P.6.a.017 Chronic ethanol exposure increases inducible nitric oxide synthase expression in the dorsolateral periaqueductal gray matter of rats

Studies about reconsolidation of conditioned fear memories have shown that pharmacological manipulation at memory reactivation can attenuate or enhance the subsequent expression of the conditioned fear response.Here we examined the effects of a single injection of the mTOR inhibitor rapamycin (Rap) into the infralimbic (IL) and prelimbic (PL) areas [which compose the ventromedial prefrontal cortex (PFC)] on reconsolidation and extinction of a traumatic fear memory.We found opposite effects of Rap infused into the PL and IL on reconsolidation and extinction: intra-PL Rap and systemic Rap impaired reconsolidation and facilitated extinction whereas intra-IL Rap enhanced reconsolidation and impaired extinction. These effects persisted at least 10 days after reactivation. Shock exposure induced anxiety-like behavior and impaired working memory and intra-IL and –PL Rap normalized these effects. Finally, when measured after fear retrieval, shocked rats exhibited reduced and increased phosphorylated p70s6K levels in the IL and basolateral amygdala, respectively. No effect on phosphorylated p70s6K levels was observed in the PL.The study points to the differential roles of the IL and PL in memory reconsolidation and extinction. Moreover, inhibiting mTOR via rapamycin following reactivation of a fear memory may be a novel approach in attenuating enhanced fear memories.

Functional and Muscular Effects of Neuromuscular Electrical Stimulation in Patients With Severe COPD: A Randomized Clinical Trial

The mechanisms through which neuromuscular electrical stimulation (NMES) training may improve limb muscle function and exercise tolerance in COPD are poorly understood. We investigated the functional and muscular effects of NMES in advanced COPD. Twenty of 22 patients with COPD were randomly assigned to NMES (n = 12) or sham (n = 8) training in a double-blind controlled study. NMES was performed on quadriceps and calf muscles, at home, 5 days per week for 6 weeks. Quadriceps and calf muscle cross-sectional area (CSA), quadriceps force and endurance, and the shuttle-walking distance with cardiorespiratory measurements were assessed before and after training. Quadriceps biopsy specimens were obtained to explore the insulin-like growth factor-1/AKT signaling pathway (70-kDa ribosomal S6 kinase [p70S6K] , atrogin-1). NMES training improved muscle CSA (P < .05), force, and endurance (P < .03) when compared with sham training. Phosphorylated p70S6K levels (anabolism) were increased after NMES as compared with sham (P = .03), whereas atrogin-1 levels (catabolism) were reduced (P = .01). Changes in quadriceps strength and ventilation during walking contributed independently to variations in walking distance after training (r = 0.77, P < .001). Gains in walking distance were related to the ability to tolerate increasing current intensities during training (r = 0.95, P < .001). In patients with severe COPD, NMES improved muscle CSA. This was associated with a more favorable muscle anabolic to catabolic balance. Improvement in walking distance after NMES training was associated with gains in muscle strength, reduced ventilation during walking, and the ability to tolerate higher stimulation intensity. ClinicalTrials.gov; No.: NCT00874965; URL: www.clinicaltrials.gov.

Heterozygous knockout of neuregulin-1 gene in mice exacerbates doxorubicin-induced heart failure

Neuregulins and their erbB receptors are essential for cardiac development and postulated to be cardioprotective in the presence of injury in the postnatal heart. We tested the hypothesis that the development of doxorubicin-induced cardiotoxicity in vivo is more severe in mice with heterozygous knockout of the neuregulin-1 gene (NRG-1(+/-)) compared with wild-type mice (WT). Three-month old NRG-1(+/-) and WT mice were injected with a single dose of doxorubicin (20 mg/kg ip). Survival was analyzed by the Kaplan-Meier approach. Left ventricular (LV) function and signaling pathways were analyzed 4 days after treatment. Fifteen days after treatment, survival was significantly lower in doxorubicin-treated NRG-1(+/-) mice (NRG-1(+/-)-Dox) compared with doxorubicin-treated WT mice (WT-Dox) (15% vs. 33%, P < 0.01). LV mass was significantly lower in NRG-1(+/-)-Dox but not in WT-Dox compared with nontreated animals. LV systolic pressure and LV midwall fractional shortening were significantly lower in NRG-1(+/-)-Dox compared with WT-Dox mice. LV protein levels of NRG-1, erbB2, and erbB4 receptors were similar in WT-Dox and NRG-1(+/-)-Dox mice. However, levels of phosphorylated erbB2, Akt, and ERK-1/2 were significantly decreased in NRG-1(+/-)-Dox compared with WT-Dox mice. A significant decrease in phosphorylated P70S6K levels was also observed in NRG-1(+/-)-Dox compared with nontreated NRG-1(+/-) mice. These results demonstrate that heterozygous knockout of the neuregulin-1 gene worsens survival and LV function in the presence of doxorubicin-induced cardiac injury in vivo. This is associated with the depression of activation of the erbB2 receptor as well as Akt, p70S6K, and ERK-1/2 pathways.