The Combination of RAD001 and NVP-BEZ235 Exerts Synergistic Anticancer Activity against Non-Small Cell Lung Cancer In Vitro and In Vivo

The phosphoinositide 3-kinase (PI3K)-mammalian target of rapamycin (mTOR) signaling axis has emerged as a novel target for cancer therapy. Agents that inhibit PI3K, mTOR or both are currently under development. The mTOR allosteric inhibitor, RAD001, and the PI3K/mTOR dual kinase inhibitor, BEZ235, are examples of these agents. We were interested in developing strategies to enhance mTOR-targeted caner therapy. In this study, we found that BEZ235 alone effectively inhibited the growth of rapamycin-resistant cancer cells. Interestingly, the combination of sub-optimal concentrations of RAD001 and BEZ235 exerted synergistic inhibition of the growth of human lung cancer cells along with induction of apoptosis and G1 arrest. Furthermore, the combination was also more effective than either agent alone in inhibiting the growth of lung cancer xenografts in mice. The combination showed enhanced effects on inhibiting mTOR signaling and reducing the expression of c-Myc and cyclin D1. Taken together, our results suggest that the combination of RAD001 and BEZ235 is a novel strategy for cancer therapy

Clinical and physiological effects of transcranial electrical stimulation position on motor evoked potentials in scoliosis surgery

<p>Abstract</p> <p>Background</p> <p>During intraoperative monitoring for scoliosis surgery, we have previously elicited ipsilateral and contralateral motor evoked potentials (MEP) with cross scalp stimulation. Ipsilateral MEPs, which may have comprised summation of early ipsilaterally conducted components and transcallosally or deep white matter stimulated components, can show larger amplitudes than those derived purely from contralateral motor cortex stimulation. We tested this hypothesis using two stimulating positions. We compared intraoperative MEPs in 14 neurologically normal subjects undergoing scoliosis surgery using total intravenous anesthetic regimens.</p> <p>Methods</p> <p>Trancranial electrical stimulation was applied with both cross scalp (C3C4 or C4C3) or midline (C3Cz or C4Cz) positions. The latter was assumed to be more focal and result in little transcallosal/deep white matter stimulation. A train of 5 square wave stimuli 0.5 ms in duration at up to 200 mA was delivered with 4 ms (250 Hz) interstimulus intervals. Averaged supramaximal MEPs were obtained from the tibialis anterior bilaterally.</p> <p>Results</p> <p>The cross scalp stimulating position resulted in supramaximal MEPs that were of significantly higher amplitude, shorter latency and required lower stimulating intensity to elicit overall (Wilcoxon Signed Rank test, p < 0.05 for all), as compared to the midline stimulating position. However, no significant differences were found for all 3 parameters comparing ipsilaterally and contralaterally recorded MEPs (p > 0.05 for all), seen for both stimulating positions individually.</p> <p>Conclusions</p> <p>Our findings suggest that cross scalp stimulation resulted in MEPs obtained ipsilaterally and contralaterally which may be contributed to by summation of ipsilateral and simultaneous transcallosally or deep white matter conducted stimulation of the opposite motor cortex. Use of this stimulating position is advocated to elicit MEPs under operative circumstances where anesthetic agents may cause suppression of cortical and spinal excitability. Although less focal in nature, cross scalp stimulation would be most suitable for infratentorial or spinal surgery, in contrast to supratentorial neurosurgical procedures.</p

c-Src Regulates Akt Signaling in Response to Ghrelin via β-Arrestin Signaling-Independent and -Dependent Mechanisms

The aim of the present study was to identify the signaling mechanisms to ghrelin-stimulated activation of the serine/threonine kinase Akt. In human embryonic kidney 293 (HEK293) cells transfected with GHS-R1a, ghrelin leads to the activation of Akt through the interplay of distinct signaling mechanisms: an early Gi/o protein-dependent pathway and a late pathway mediated by β-arrestins. The starting point is the Gi/o-protein dependent PI3K activation that leads to the membrane recruitment of Akt, which is phosphorylated at Y by c-Src with the subsequent phosphorylation at A-loop (T308) and HM (S473) by PDK1 and mTORC2, respectively. Once the receptor is activated, a second signaling pathway is mediated by β-arrestins 1 and 2, involving the recruitment of at least β-arrestins, c-Src and Akt. This β-arrestin-scaffolded complex leads to full activation of Akt through PDK1 and mTORC2, which are not associated to the complex. In agreement with these results, assays performed in 3T3-L1 preadipocyte cells indicate that β-arrestins and c-Src are implicated in the activation of Akt in response to ghrelin through the GHS-R1a. In summary this work reveals that c-Src is crucially involved in the ghrelin-mediated Akt activation. Furthermore, the results support the view that β-arrestins act as both scaffolding proteins and signal transducers on Akt activation

The role of mTOR and phospho-p70S6K in pathogenesis and progression of gastric carcinomas: an immunohistochemical study on tissue microarray

<p>Abstract</p> <p>Background</p> <p>mTOR signaling pathway and its downstream serine/threonine kinase p70S6k were frequently activated in human cancers. The dysregulation of the mTOR pathway has been found to be a contributing factor of a variety of different cancer. To investigate the role of mTOR signal pathway in the stepwise development of gastric carcinomas, we analyzed the correlations between the mTOR and P70S6K expression and clinic pathological factors and studied its prognostic role in gastric carcinomas.</p> <p>Methods</p> <p>mTOR and phospho-p70S6K proteins were examined by immunohistochemistry on tissue microarray containing gastric carcinomas (n = 412), adenomas (n = 47) and non-neoplastic mucosa (NNM, n = 197) with a comparison of their expression with clinicopathological parameters of carcinomas.</p> <p>Results</p> <p>There was no difference of mTOR expression between these three tissues (p > 0.05). Cytoplasmic phospho(p)-P706SK was highly expressed in adenoma, compared with ANNMs (p < 0.05), whereas its nuclear expression was lower in gastric carcinomas than gastric adenoma and ANNMs (p < 0.05). These three markers were preferably expressed in the older patients with gastric cancer and intestinal-type carcinoma (p < 0.05). mTOR expression was positively correlated with the cytoplasmic and nuclear expression of p-P70S6K(p < 0.05). Nuclear P70S6K was inversely linked to tumor size, depth of invasion, lymph node metastasis and UICC staging (p < 0.05). Univariate analysis indicated that expression of mTOR and nuclear p-P70S6K was closely linked to favorable prognosis of the carcinoma patients (p < 0.05). Multivariate analysis showed that age, depth of invasion, lymphatic invasion, lymph node metastasis, Lauren's classification and mTOR expression were independent prognostic factors for overall gastric carcinomas (p < 0.05).</p> <p>Conclusion</p> <p>Aberrant expression of p-P70S6K possibly contributes to pathogenesis, growth, invasion and metastasis of gastric carcinomas. It was considered as a promising marker to indicate the aggressive behaviors and prognosis of gastric carcinomas.</p

Azimuthal anisotropy and correlations at large transverse momenta in p+pp+p and Au+Au collisions at sNN\sqrt{s_{_{NN}}}= 200 GeV

Results on high transverse momentum charged particle emission with respect to the reaction plane are presented for Au+Au collisions at $\sqrt{s_{_{NN}}}$= 200 GeV. Two- and four-particle correlations results are presented as well as a comparison of azimuthal correlations in Au+Au collisions to those in $p+p$ at the same energy. Elliptic anisotropy, $v_2$, is found to reach its maximum at $p_t \sim 3$ GeV/c, then decrease slowly and remain significant up to $p_t\approx 7$ -- 10 GeV/c. Stronger suppression is found in the back-to-back high-$p_t$ particle correlations for particles emitted out-of-plane compared to those emitted in-plane. The centrality dependence of $v_2$ at intermediate $p_t$ is compared to simple models based on jet quenching.Comment: 4 figures. Published version as PRL 93, 252301 (2004

Azimuthal anisotropy in Au+Au collisions at sqrtsNN = 200 GeV

The results from the STAR Collaboration on directed flow (v_1), elliptic flow (v_2), and the fourth harmonic (v_4) in the anisotropic azimuthal distribution of particles from Au+Au collisions at sqrtsNN = 200 GeV are summarized and compared with results from other experiments and theoretical models. Results for identified particles are presented and fit with a Blast Wave model. Different anisotropic flow analysis methods are compared and nonflow effects are extracted from the data. For v_2, scaling with the number of constituent quarks and parton coalescence is discussed. For v_4, scaling with v_2^2 and quark coalescence is discussed.Comment: 26 pages. As accepted by Phys. Rev. C. Text rearranged, figures modified, but data the same. However, in Fig. 35 the hydro calculations are corrected in this version. The data tables are available at http://www.star.bnl.gov/central/publications/ by searching for "flow" and then this pape

Rapidity and Centrality Dependence of Proton and Anti-proton Production from Au+Au Collisions at sqrt(sNN) = 130GeV

We report on the rapidity and centrality dependence of proton and anti-proton transverse mass distributions from Au+Au collisions at sqrt(sNN) = 130GeV as measured by the STAR experiment at RHIC. Our results are from the rapidity and transverse momentum range of |y|<0.5 and 0.35 <p_t<1.00GeV/c. For both protons and anti-protons, transverse mass distributions become more convex from peripheral to central collisions demonstrating characteristics of collective expansion. The measured rapidity distributions and the mean transverse momenta versus rapidity are flat within |y|<0.5. Comparisons of our data with results from model calculations indicate that in order to obtain a consistent picture of the proton(anti-proton) yields and transverse mass distributions the possibility of pre-hadronic collective expansion may have to be taken into account.Comment: 4 pages, 3 figures, 1 table, submitted to PR

The Drosophila FoxA Ortholog Fork Head Regulates Growth and Gene Expression Downstream of Target of Rapamycin

Forkhead transcription factors of the FoxO subfamily regulate gene expression programs downstream of the insulin signaling network. It is less clear which proteins mediate transcriptional control exerted by Target of rapamycin (TOR) signaling, but recent studies in nematodes suggest a role for FoxA transcription factors downstream of TOR. In this study we present evidence that outlines a similar connection in Drosophila, in which the FoxA protein Fork head (FKH) regulates cellular and organismal size downstream of TOR. We find that ectopic expression and targeted knockdown of FKH in larval tissues elicits different size phenotypes depending on nutrient state and TOR signaling levels. FKH overexpression has a negative effect on growth under fed conditions, and this phenotype is not further exacerbated by inhibition of TOR via rapamycin feeding. Under conditions of starvation or low TOR signaling levels, knockdown of FKH attenuates the size reduction associated with these conditions. Subcellular localization of endogenous FKH protein is shifted from predominantly cytoplasmic on a high-protein diet to a pronounced nuclear accumulation in animals with reduced levels of TOR or fed with rapamycin. Two putative FKH target genes, CG6770 and cabut, are transcriptionally induced by rapamycin or FKH expression, and silenced by FKH knockdown. Induction of both target genes in heterozygous TOR mutant animals is suppressed by mutations in fkh. Furthermore, TOR signaling levels and FKH impact on transcription of the dFOXO target gene d4E-BP, implying a point of crosstalk with the insulin pathway. In summary, our observations show that an alteration of FKH levels has an effect on cellular and organismal size, and that FKH function is required for the growth inhibition and target gene induction caused by low TOR signaling levels

Resveratrol Inhibits Protein Translation in Hepatic Cells

Resveratrol is a plant-derived polyphenol that extends lifespan and healthspan in model organism. Despite extensive investigation, the biological processes mediating resveratrol's effects have yet to be elucidated. Because repression of translation shares many of resveratrol's beneficial effects, we hypothesized that resveratrol was a modulator of protein synthesis. We studied the effect of the drug on the H4-II-E rat hepatoma cell line. Initial studies showed that resveratrol inhibited global protein synthesis. Given the role of the mammalian Target of Rapamycin (mTOR) in regulating protein synthesis, we examined the effect of resveratrol on mTOR signaling. Resveratrol inhibited mTOR self-phosphorylation and the phosphorylation of mTOR targets S6K1 and eIF4E-BP1. It attenuated the formation of the translation initiation complex eIF4F and increased the phosphorylation of eIF2α. The latter event, also a mechanism for translation inhibition, was not recapitulated by mTOR inhibitors. The effects on mTOR signaling were independent of effects on AMP-activated kinase or AKT. We conclude that resveratrol is an inhibitor of global protein synthesis, and that this effect is mediated through modulation of mTOR-dependent and independent signaling

Rapamycin Pharmacokinetic and Pharmacodynamic Relationships in Osteosarcoma: A Comparative Oncology Study in Dogs

Signaling through the mTOR pathway contributes to growth, progression and chemoresistance of several cancers. Accordingly, inhibitors have been developed as potentially valuable therapeutics. Their optimal development requires consideration of dose, regimen, biomarkers and a rationale for their use in combination with other agents. Using the infrastructure of the Comparative Oncology Trials Consortium many of these complex questions were asked within a relevant population of dogs with osteosarcoma to inform the development of mTOR inhibitors for future use in pediatric osteosarcoma patients.This prospective dose escalation study of a parenteral formulation of rapamycin sought to define a safe, pharmacokinetically relevant, and pharmacodynamically active dose of rapamycin in dogs with appendicular osteosarcoma. Dogs entered into dose cohorts consisting of 3 dogs/cohort. Dogs underwent a pre-treatment tumor biopsy and collection of baseline PBMC. Dogs received a single intramuscular dose of rapamycin and underwent 48-hour whole blood pharmacokinetic sampling. Additionally, daily intramuscular doses of rapamycin were administered for 7 days with blood rapamycin trough levels collected on Day 8, 9 and 15. At Day 8 post-treatment collection of tumor and PBMC were obtained. No maximally tolerated dose of rapamycin was attained through escalation to the maximal planned dose of 0.08 mg/kg (2.5 mg/30 kg dog). Pharmacokinetic analysis revealed a dose-dependent exposure. In all cohorts modulation of the mTOR pathway in tumor and PBMC (pS6RP/S6RP) was demonstrated. No change in pAKT/AKT was seen in tumor samples following rapamycin therapy.Rapamycin may be safely administered to dogs and can yield therapeutic exposures. Modulation pS6RP/S6RP in tumor tissue and PBMCs was not dependent on dose. Results from this study confirm that the dog may be included in the translational development of rapamycin and potentially other mTOR inhibitors. Ongoing studies of rapamycin in dogs will define optimal schedules for their use in cancer and evaluate the role of rapamycin use in the setting of minimal residual disease