NVP-BEZ235 Inhibits Renal Cell Carcinoma by Targeting TAK1 and PI3K/Akt/mTOR Pathways

In spite of the promising in vitro and preclinical results, dual PI3K/Akt/mTOR inhibitor NVP-BEZ235, and ATP-competitive mTOR inhibitor PP242 both failed to confirm their inhibitory efficacy against renal cell carcinoma (RCC) in clinical settings. Therefore, a better understanding of the molecular mechanism is essential so as to provide possibilities for their use in combination with other agents. In present study, RCC cell lines (UMRC6, 786-0 and UOK121) were treated with NVP-BEZ235, PP242 or Rapamycin, an mTOR complex 1 (mTORC1)-specific inhibitor. They all suppressed cell proliferation and invasion, induced apoptosis and cell cycle arrest, and the effects were in the order of NVP-BEZ235 > PP242 > Rapamycin. Accordingly, the marked and sustained decrease in speckle-type POZ protein (SPOP) expression and phosphorylation of Akt and mTOR kinases was observed in RCC cells treated with NVP-BEZ235 and PP242, whereas only potent inhibition of mTOR activity was induced in Rapamycin-treated cells. In considering the overactivation of c-Jun and IκB-α in human renal tumor tissue, we next investigated the role of JNK and IKK pathways in the response of RCC cells to these compounds. First of all, transforming growth factor β activated kinase 1 (TAK1)-dependent activation of JNK/ (activator protein-1) AP-1 axis in RCC cells was proved by the repression of AP-1 activity with TAK1 or JNK inhibitor. Second, the profound inhibition of TAK1/JNK/AP-1 pathway was demonstrated in RCC cells treated with NVP-BEZ235 or PP242 but not Rapamycin, which is manifested as a reduction in activity of TAK1, c-Jun and AP-1. Meanwhile, subsequent to TAK1 inactivation, the activation of IκB-α was also reduced by NVP-BEZ235 and PP242. Likewise, in vivo, treatment with NVP-BEZ235 and PP242 suppressed the growth of xenografts generated from 786-0 and A498 cells, along with decreased expression of phospho-TAK1, phospho-c-Jun, and phospho-IκB-α. In contrast, Rapamycin elicited no significant inhibitory effects on tumor growth and phosphorylation of TAK1, c-Jun and IκB-α. We conclude that besides PI3K/Akt/mTOR signaling, NVP-BEZ235, and PP242 simultaneously target TAK1-dependent pathways in RCC cells. Notably, these effects were more marked in the presence of NVP-BEZ235 than PP242, indicating the potential application of NVP-BEZ235 in combination therapy for RCC.

ROLE OF ΚB AND AP-1 TRANSCRIPTION FACTORS IN CHANGES IN NITRIC OXIDE PRODUCTION AND UTILIZATION IN THE HEART OF RATS UNDER CHRONIC FLUORIDE INTOXICATION

Fluorides, being hazardous contaminants of soil and drinking water, can get in excessive amounts into human and animal bodies. This is especially true for regions where the fluoride content in soils is very high, for example, Poltava, Dnipropetrovsk, and Kirovohrad regions in Ukraine. Excessive fluoride intake can change the rate of nitric oxide production. The impact of fluorides on changes in nitric oxide production and metabolism in the heart and the role of redox-sensitive transcription factors in these changes are poorly understood. The aim of this study was to determine the effect of activation of κB transcription factors and activator protein 1 on the activity of inducible NO-synthase, constitutive isoforms of NO-synthase, nitrite and nitrate reductase, arginase, concentration of nitrites, peroxynitrite and nitrosothiols in the heart of rats during chronic fluoride intoxication. Materials and methods. The study was performed on 24 adult male Wistar rats weighing 220-260 grams. Animals were randomly divided into 4 groups of 6 animals in each (control, chronic fluoride intoxication group, κB blockade group and activator protein 1 blockade group). The experiment lasted 30 days. We determined the activity of inducible NO-synthase, constitutive isoforms of NO-synthase, the concentration of peroxynitrite alkali and alkaline earth metals, the concentration of nitrites and nitrosothiols, the activity of nitrite reductase, nitrate reductase and arginase. Results. Chronic fluoride intoxication increases the activity of inducible NO-synthase by 1.74 times, does not affect the activity of constitutive isoforms and reduces the activity of arginase by 35.68% compared with the control group of animals. The concentration of nitrites in the heart of rats increases 1.73 times, peroxynitrite 1.43 times, and the concentration of nitrosothiols doubled. The use of κB transcription factor blockers and activator protein 1 reduces nitric oxide production from NO synthases and reduces the concentrations of all nitric oxide metabolites in the heart of rats under conditions of chronic fluoride intoxication. Conclusions. Activation of κB transcription factors and activator protein 1 during chronic excessive intake of fluoride leads to hyperproduction of nitric oxide in the heart of rats due to increased activity of inducible NO-synthase and nitrite reductases. Excess production of nitric oxide under chronic fluoride intoxication leads to the accumulation of nitrites, peroxynitrite and nitrosothiols in the heart of rats.

Fukutin Protein Participates in Cell Proliferation by Enhancing Cyclin D1 Expression through Binding to the Transcription Factor Activator Protein-1: An In Vitro Study

The causative gene of Fukuyama congenital muscular dystrophy (fukutin) is involved in formation of the basement membrane through glycosylation of alpha-dystroglycan. However, there are other proposed functions that have not been fully understood. Using cultured astrocytes (1321N1), we found nuclear localization of fukutin and a positive relationship between fukutin expression and cell proliferation. Among potential proteins regulating cell proliferation, we focused on cyclin D1, by reverse-transcription polymerase chain reaction, Western blotting, immunocytochemistry, enzyme-linked immunosorbent assay (ELISA), and sandwich ELISA. Expression of cyclin D1 was significantly downregulated by fukutin knockdown and significantly upregulated by fukutin overexpression. Moreover, fukutin was proven to bind to the activator protein-1 (AP-1) binding site of cyclin D1 promoter, as well as the AP-1 component c-Jun. The c-Jun phosphorylation status was not significantly influenced by knockdown or overexpression of fukutin. The present results provide in vitro evidence for a novel function of fukutin, which participates in cell proliferation by enhancing cyclin D1 expression through forming a complex with AP-1. It is likely that fukutin is a potential cofactor of AP-1.

Early Pro-Inflammatory Signal and T-Cell Activation Associate With Vaccine-Induced Anti-Vaccinia Protective Neutralizing Antibodies

Both vaccine “take” and neutralizing antibody (nAb) titer are historical correlates for vaccine-induced protection from smallpox. We analyzed a subset of samples from a phase 2a trial of three DNA/HIV-1 primes and a recombinant Tiantan vaccinia virus-vectored (rTV)/HIV-1 booster and found that a proportion of participants showed no anti-vaccinia nAb response to the rTV/HIV-1 booster, despite successful vaccine “take.” Using a rich transcriptomic and vaccinia-specific immunological dataset with fine kinetic sampling, we investigated the molecular mechanisms underlying nAb response. Blood transcription module analysis revealed the downregulation of the activator protein 1 (AP-1) pathway in responders, but not in non-responders, and the upregulation of T-cell activation in responders. Furthermore, transcriptional factor network reconstruction revealed the upregulation of AP-1 core genes at hour 4 and day 1 post-rTV/HIV-1 vaccination, followed by a downregulation from day 3 until day 28 in responders. In contrast, AP-1 core and pro-inflammatory genes were upregulated on day 7 in non-responders. We speculate that persistent pro-inflammatory signaling early post-rTV/HIV-1 vaccination inhibits the nAb response.

The Ascidia Ciona robusta Provides Novel Insights on the Evolution of the AP-1 Transcriptional Complex

The Activator Protein-1 transcription factor family (AP-1) transcriptional complex is historically defined as an early response group of transcription factors formed by dimeric complexes of the Jun, Fos, Atf, and Maf bZIP proteins that control cell proliferation and differentiation by regulating gene expression. It has been greatly investigated in many model organisms across metazoan evolution. Nevertheless, its complexity and variability of action made its multiple functions difficult to be defined. Here, we place the foundations for understanding the complexity of AP-1 transcriptional members in tunicates. We investigated the gene members of this family in the ascidian Ciona robusta and identified single copies of Jun, Fos, Atf3, Atf2/7, and Maf bZIP-related factors that could have a role in the formation of the AP-1 complex. We highlight that mesenchyme is a common cellular population where all these factors are expressed during embryonic development, and that, moreover, Fos shows a wider pattern of expression including also notochord and neural cells. By ectopic expression in transgenic embryos of Jun and Fos genes alone or in combination, we investigated the phenotypic alterations induced by these factors and highlighted a degree of functional conservation of the AP-1 complex between Ciona and vertebrates. The lack of gene redundancy and the first pieces of evidence of conserved functions in the control of cell movements and structural organization exerted by these factors open the way for using Ciona as a helpful model system to uncover the multiple potentialities of this highly complex family of bZIP transcription factors.