NFAT5 Is Up-Regulated by Hypoxia: Possible Implications in Preeclampsia and Intrauterine Growth Restriction.

During gestation, low oxygen environment is a major determinant of early placentation process, while persistent placental hypoxia leads to pregnancy-related complications such as preeclampsia (PE) and intrauterine growth restriction (IUGR). PE affects 5%-8% of all pregnancies worldwide and is a cause of maternal and fetal morbidity and mortality. During placental development, persistent hypoxia due to poor trophoblast invasion and reduced uteroplacental perfusion leads to maternal endothelial dysfunction and clinical manifestation of PE. Here we hypothesized that nuclear factor of activated T cells-5 (NFAT5), a well-known osmosensitive renal factor and recently characterized hypoxia-inducible protein, is also activated in vivo in placentas of PE and IUGR complications as well as in the in vitro model of trophoblast hypoxia. In JAR cells, low oxygen tension (1% O2) induced NFAT5 mRNA and increased its nuclear abundance, peaking at 16 h. This increase did not occur in parallel with the earlier HIF1A induction. Real-time PCR and Western blot analysis confirmed up-regulation of NFAT5 mRNA and NFAT5 nuclear content in human preeclamptic placentas and in rabbit placentas of an experimentally induced IUGR model, as compared with the control groups. In vitro lambda protein phosphatase (lambda PPase) treatment revealed that increased abundance of NFAT5 protein in nuclei of either JAR cells (16 h of hypoxia) or PE and IUGR placentas is at least partially due to NFAT5 phosphorylation. NFAT5 downstream targets aldose reductase (AR) and sodium-myo-inositol cotransporter (SMIT; official symbol SLC5A3) were not significantly up-regulated either in JAR cells exposed to hypoxia or in placentas of PE- and IUGR-complicated pregnancies, suggesting that hypoxia-dependent activation of NFAT5 serves as a separate function to its tonicity-dependent stimulation. In conclusion, we propose that NFAT5 may serve as a novel marker of placental hypoxia and ischemia independently of HIF1A.

The STAT3 inhibitor GPB730 enhances the sensitivity to enzalutamide in prostate cancer cells.

Enzalutamide is a second-generation anti-androgen which has shown increased survival in patients with metastatic prostate cancer. However, some patients do not respond to this therapy or will develop resistance to treatment over time. Signal Transducer and Activator of Transcription 3 (STAT3) is known to be involved in castration-resistant prostate cancer and to interact with androgen receptor (AR)-signaling. This study aims to investigate the combination enzalutamide and the small molecule STAT3 inhibitor GPB730 for enhanced therapeutic effect in advanced prostate cancer in vitro. The prostate cancer cell lines LNCaP (androgen dependent) and C4-2 (androgen insensitive) were used. The effect of enzalutamide and GPB730, alone and in combination, was investigated on viability and IC50 values calculated. Enzalutamide and GPB730 treated LNCaP and C4-2 cells were subjected to western blot and QPCR analyses in order to investigate the expression of AR, STAT3 and down-stream targets. C4-2 were less sensitive to growth inhibition by enzalutamide than LNCaP cells. GPB730 enhanced the growth inhibitory effect of enzalutamide in LNCaP and C4-2 cells. The addition of GPB730 to enzalutamide decreased the IC50 values for enzalutamide by 3.3-fold for LNCaP and by 12-fold for C4-2. In C4-2 cells, GPB730 alone decreased PSA expression and enhanced the enzalutamide induced decrease in NKX3.1 expression. GPB730 and enzalutamide in combination enhanced inhibition of c-myc and survivin expression. This study suggests that enzalutamide may be combined with the STAT3 inhibitor GPB730 in order to enhance the efficacy of enzalutamide, offering a new therapeutic approach in advanced prostate cancer.

STAT3 inhibition with galiellalactone effectively targets the prostate cancer stem-like cell population.

Cancer stem cells (CSCs) are a small subpopulation of quiescent cells with the potential to differentiate into tumor cells. CSCs are involved in tumor initiation and progression and contribute to treatment failure through their intrinsic resistance to chemo- or radiotherapy, thus representing a substantial concern for cancer treatment. Prostate CSCs' activity has been shown to be regulated by the transcription factor Signal Transducer and Activator of Transcription 3 (STAT3). Here we investigated the effect of galiellalactone (GL), a direct STAT3 inhibitor, on CSCs derived from prostate cancer patients, on docetaxel-resistant spheres with stem cell characteristics, on CSCs obtained from the DU145 cell line in vitro and on DU145 tumors in vivo. We found that GL significantly reduced the viability of docetaxel-resistant and patient-derived spheres. Moreover, CSCs isolated from DU145 cells were sensitive to low concentrations of GL, and the treatment with GL suppressed their viability and their ability to form colonies and spheres. STAT3 inhibition down regulated transcriptional targets of STAT3 in these cells, indicating STAT3 activity in CSCs. Our results indicate that GL can target the prostate stem cell niche in patient-derived cells, in docetaxel-resistant spheres and in an in vitro model. We conclude that GL represents a promising therapeutic approach for prostate cancer patients, as it reduces the viability of prostate cancer-therapy-resistant cells in both CSCs and non-CSC populations.

Scavenging of Labile Heme by Hemopexin Is a Key Checkpoint in Cancer Growth and Metastases.

Hemopexin (Hx) is a scavenger of labile heme. Herein, we present data defining the role of tumor stroma-expressed Hx in suppressing cancer progression. Labile heme and Hx levels are inversely correlated in the plasma of patients with prostate cancer (PCa). Further, low expression of Hx in PCa biopsies characterizes poorly differentiated tumors and correlates with earlier time to relapse. Significantly, heme promotes tumor growth and metastases in an orthotopic murine model of PCa, with the most aggressive phenotype detected in mice lacking Hx. Mechanistically, labile heme accumulates in the nucleus and modulates specific gene expression via interacting with guanine quadruplex (G4) DNA structures to promote PCa growth. We identify c-MYC as a heme:G4-regulated gene and a major player in heme-driven cancer progression. Collectively, these results reveal that sequestration of labile heme by Hx may block heme-driven tumor growth and metastases, suggesting a potential strategy to prevent and/or arrest cancer dissemination.

Impairment of Angiogenic Sphingosine Kinase-1/Sphingosine-1-Phosphate Receptors Pathway in Preeclampsia.

Preeclampsia (PE), is a serious pregnancy disorder characterized in the early gestation by shallow trophoblast invasion, impaired placental neo-angiogenesis, placental hypoxia and ischemia, which leads to maternal and fetal morbidity and mortality. Here we hypothesized that angiogenic sphingosine kinase-1 (SPHK1)/sphingosine-1-phosphate (S1P) receptors pathway is impaired in PE. We found that SPHK1 mRNA and protein expression are down-regulated in term placentae and term chorionic villous explants from patients with PE or severe PE (PES), compared with controls. Moreover, mRNA expression of angiogenic S1PR1 and S1PR3 receptors were decreased in placental samples of PE and PES patients, whereas anti-angiogenic S1PR2 was up-regulated in chorionic villous tissue of PES subjects, pointing to its potential atherogenic and inflammatory properties. Furthermore, in in vitro (JAR cells) and ex vivo (chorionic villous explants) models of placental hypoxia, SPHK1 mRNA and protein were strongly up-regulated under low oxygen tension (1% 02). In contrast, there was no change in SPHK1 expression under the conditions of placental physiological hypoxia (8% 02). In both models, nuclear protein levels of HIF1A were increased at 1% 02 during the time course, but there was no up-regulation at 8% 02, suggesting that SPHK1 and HIF1A might be the part of the same canonical pathway during hypoxia and that both contribute to placental neovascularization during early gestation. Taken together, this study suggest the SPHK1 pathway may play a role in the human early placentation process and may be involved in the pathogenesis of PE.

Efecto de la terapia de resincronización ventricular en los parámetros de disfunción endotelial y función sistólica izquierda en pacientes con insuficiencia cardíaca crónica y bloqueo completo de rama izquierda / Endothelial dysfunction, left ventricular function, and chronic heart failure with complete left bundle branch block: effect of resynchronization therapy

Resumen: Introducción: La insuficiencia cardíaca crónica (ICC) es una condición compleja asociada a inflamación sistémica y a disfunción endotelial (DE) cuya patogénesis no es bien comprendida. Objetivo: Evaluar una posible relación entre marcadores de DE periférica con la respuesta a terapia de resincronización ventricular (TRV). Método: 20 pacientes con ICC, QRS ≥120ms y fracción de eyección ventricular izquierda (FEVI) ≤35% se estudiaron pre y 6 meses post-TRV con: Minnesota Living with Heart Failure Questionnaire (MLHFQ); test de marcha (TM-6min); Ecocardiografía-2D y SPECT de perfusión gatillado en reposo; proteína C-reactiva ultra sensible (us-PCR); péptido natriurético cerebral (pro-BNP); células endoteliales circulantes (CEC); moléculas de adhesión soluble vascular (sVCAM) e intercelular (sICAM); interleukina-6 (IL-6) y Factor von Willebrand (FvW). Se clasificaron como respondedores o no a TRV según criterios preestablecidos. Resultados: Promedios basales: pro-BNP 5.290pg/ml; us-PCR 1,7ug/mL; MLHFQ 72; TM-6min 391 metros. Las CEC y sICAM estaban sobre límites normales. Post-TRV, el 50% fue respondedor: 11/20 mejoraron ≥1 clase NYHA y ≥10% del TM-6min; ML-HFQ disminuyó (p<0.0001); FEVI mejoró (p=0.003); volumen final sistólico disminuyó (p=0.008) y también pro-BNP (p=0.03). En los respondedores, las CEC disminuyeron, persistiendo elevadas, sobre lo normal. Existieron correlaciones entre cambios de pro-BNP con TM-6min y entre us-PCR con MLHFQ y FvW (p≤0.004 en todas). Conclusiones: En ICC existe evidencia de significativa DE, expresada por sICAM y CEC, biomarcador periférico sensible. Estas disminuyeron 6 meses post-TRV, persistiendo sobre el límite normal. Otros parámetros funcionales e inflamatorios se correlacionaron en el grupo total, sin diferencias entre grupos respondedores y no respondedores.

Abstract: Introduction: Chronic heart failure (CHF) is a complex condition associated with systemic inflammation and endothelial dysfunction (ED) whose pathogenesis is not well understood. Objective: to evaluate a possible relationship between peripheral ED markers and response to cardiac resynchronization therapy (CRT). Method: 20 patients with CHF, QRS ≥120ms and left ventricular ejection fraction (LVEF) ≤35% were studied before and 6 months post-CRT. Minnesota Living with Heart Failure Questionnaire (MLHFQ); walking test (6min-WT); 2D-echocardiography and gated perfusion SPECT at rest; ultra-sensitive C-reactive protein (us-CRP); brain natriuretic peptide (pro-BNP); circulating endothelial cells (CEC); vascular soluble adhesion (sVCAM) and intercellular adhesion molecules (sICAM); interleukin-6 (IL-6) and von Willebrand Factor (vWF) were measured in all subjects. They were classified as responders or not to CRT, according to pre-established criteria. Results: Basal means: pro-BNP 5,290 pg / ml; us-CRP 1.7 ug/mL; MLHFQ 72; 6min-WT 391 meters. The CEC and IL-6 were above normal limits. Post-CRT, 50% were responders: 11/20 improved ≥1 NYHA class and ≥10% increase in 6min-WT; MLHFQ decreased (p <0.0001); LVEF improved (p = 0.003); final systolic volume decreased (p = 0.008) and also pro-BNP (p= 0.03). In responders CEC decreased, persisting over normal limits. There were correlations between changes of pro-BNP with TM-6min and between us-PCR with MLHFQ and vWF (p≤0.004 in all). Conclusions: In CHF there is evidence of significant ED, expressed by sICAM and CEC, a sensitive peripheral biomarker that decreased 6 months post-CRT although persisting above normal limits. Other functional and inflammatory parameters were correlated in the total group, without differences between responders and non-responders.