Rottlerin Inhibits ROS Formation and Prevents NFκB Activation in MCF-7 and HT-29 Cells

Rottlerin, a polyphenol isolated from Mallotus Philippinensis, has been recently used as a selective inhibitor of PKC δ, although it can inhibit many kinases and has several biological effects. Among them, we recently found that Rottlerin inhibits the Nuclear Factor κB (NFκB), activated by either phorbol esters or H2O2. Because of the redox sensitivity of NFκB and on the basis of Rottlerin antioxidant property, we hypothesized that Rottlerin could prevent NFκB activation acting as a free radicals scavenger, as other natural polyphenols. The current study confirms the antioxidant property of Rottlerin against the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) in vitro and against oxidative stress induced by H2O2 and by menadione in culture cells. We also demonstrate that Rottlerin prevents TNFα-dependent NFκB activation in MCF-7 cells and in HT-29 cells transfected with the NFκB-driven plasmid pBIIX-LUC, suggesting that Rottlerin can inhibit NFκB via several pathways and in several cell types

eIF4F controls ERK MAPK signaling in melanomas with BRAF and NRAS mutations

The eIF4F translaon iniaon complex plays a crical role in melanoma resistance to clinical BRAF and MEK inhibitors. In this study, we uncover a novel funcon of eIF4F in the negave regulaon of the RAS/RAF/MEK/ERK mitogen-acvated protein kinase (MAPK) signaling pathway. We demonstrate that eIF4F is essenal for controlling ERK signaling intensity in treatment-naïve melanoma cells harboring BRAF or NRAS mutaons. Specifically, the dual-specificity phosphatase DUSP6/MKP3, which acts as a negave feedback regulator of ERK acvity, requires connuous producon in an eIF4F-dependent manner to limit excessive ERK signaling driven by oncogenic RAF/RAS mutaons. Treatment with small molecule eIF4F inhibitorsdisrupts the negave feedback control of MAPK signaling, leading to ERK hyperacvaon and EGR1 overexpression in melanoma cells in vitro and in vivo. Furthermore, our quantave analyses reveal a high spare signaling capacity in the ERK pathway, suggesng that eIF4F-dependent feedback keeps the majority of ERK molecules inacve under normal condions. Overall, our findings highlight the crucial role of eIF4F in regulang ERK signaling flux and suggest that pharmacological eIF4F inhibitors can disrupt the negave feedback control of MAPK acvity in melanomas with BRAF and NRAS acvang mutaons

Dynamic Monitoring of Cellular Remodeling Induced by the Transforming Growth Factor-β1

The plasticity of differentiated adult cells could have a great therapeutic potential, but at the same time, it is characteristic of progression of serious pathological states such as cancer and fibrosis. In this study, we report on the application of a real-time noninvasive system for dynamic monitoring of cellular plasticity. Analysis of the cell impedance profile recorded as cell index using a real-time cell analyzer revealed its significant increase after the treatment of prostate epithelial cells with the transforming growth factor-β1. Changes in the cell index profile were paralleled with cytoskeleton rebuilding and induction of epithelial–mesenchymal transition and negatively correlated with cell proliferation. This novel application of such approach demonstrated a great potential of the impedance-based system for noninvasive and real-time monitoring of cellular fate

Pre-existing cell subpopulations in primary prostate cancer tumors display surface fingerprints of docetaxel-resistant cells

PurposeDocetaxel resistance is a significant obstacle in the treatment of prostate cancer (PCa), resulting in unfavorable patient prognoses. Intratumoral heterogeneity, often associated with epithelial-to-mesenchymal transition (EMT), has previously emerged as a phenomenon that facilitates adaptation to various stimuli, thus promoting cancer cell diversity and eventually resistance to chemotherapy, including docetaxel. Hence, understanding intratumoral heterogeneity is essential for better patient prognosis and the development of personalized treatment strategies.MethodsTo address this, we employed a high-throughput single-cell flow cytometry approach to identify a specific surface fingerprint associated with docetaxel-resistance in PCa cells and complemented it with proteomic analysis of extracellular vesicles. We further validated selected antigens using docetaxel-resistant patient-derived xenografts in vivo and probed primary PCa specimens to interrogate of their surface fingerprint.ResultsOur approaches revealed a 6-molecule surface fingerprint linked to docetaxel resistance in primary PCa specimens. We observed consistent overexpression of CD95 (FAS/APO-1), and SSEA-4 surface antigens in both in vitro and in vivo docetaxel-resistant models, which was also observed in a cell subpopulation of primary PCa tumors exhibiting EMT features. Furthermore, CD95, along with the essential enzymes involved in SSEA-4 synthesis, ST3GAL1, and ST3GAL2, displayed a significant increase in patients with PCa undergoing docetaxel-based therapy, correlating with poor survival outcomes.ConclusionIn summary, we demonstrate that the identified 6-molecule surface fingerprint associated with docetaxel resistance pre-exists in a subpopulation of primary PCa tumors before docetaxel treatment. Thus, this fingerprint warrants further validation as a promising predictive tool for docetaxel resistance in PCa patients prior to therapy initiation

Novel CHK1 inhibitor MU380 exhibits significant single-agent activity in TP53-mutated chronic lymphocytic leukemia cells

Introduction of small-molecule inhibitors of B-cell receptor signaling and BCL2 protein significantly improves therapeutic options in chronic lymphocytic leukemia. However, some patients suffer from adverse effects mandating treatment discontinuation, and cases with TP53 defects more frequently experience early progression of the disease. Development of alternative therapeutic approaches is, therefore, of critical importance. Here we report details of the anti-chronic lymphocytic leukemia single-agent activity of MU380, our recently identified potent, selective, and metabolically robust inhibitor of checkpoint kinase 1. We also describe a newly developed enantioselective synthesis of MU380, which allows preparation of gram quantities of the substance. Checkpoint kinase 1 is a master regulator of replication operating primarily in intra-S and G2/M cell cycle checkpoints. Initially tested in leukemia and lymphoma cell lines, MU380 significantly potentiated efficacy of gemcitabine, a clinically used inducer of replication stress. Moreover, MU380 manifested substantial single-agent activity in both TP53-wild type and TP53-mutated leukemia and lymphoma cell lines. In chronic lymphocytic leukemia-derived cell lines MEC-1, MEC-2 (both TP53-mut), and OSU-CLL (TP53-wt) the inhibitor impaired cell cycle progression and induced apoptosis. In primary clinical samples, MU380 used as a single-agent noticeably reduced the viability of unstimulated chronic lymphocytic leukemia cells as well as those induced to proliferate by anti-CD40/IL-4 stimuli. In both cases, effects were comparable in samples harboring p53 pathway dysfunction (TP53 mutations or ATM mutations) and TP53-wt/ATM-wt cells. Lastly, MU380 also exhibited significant in vivo activity in a xenotransplant mouse model (immunodeficient strain NOD-scid IL2Rγnull) where it efficiently suppressed growth of subcutaneous tumors generated from MEC-1 cells

3D cell culture models demonstrate a role for FGF and WNT signaling in regulation of lung epithelial cell fate and morphogenesis

AbstractFGF signaling plays an essential role in lung development, homeostasis, and regeneration. Several FGF ligands were detected in the developing lungs, however, their roles have not been fully elucidated. We employed mouse 3D cell culture models and imaging toex vivostudy ofa)the role of FGF ligands in lung epithelial morphogenesis andb)the interplay of FGF signaling with epithelial growth factor (EGF) and WNT signaling pathways. In non-adherent conditions, FGF signaling promoted formation of lungospheres from lung epithelial stem/progenitor cells (LSPCs). Based on their architecture, we defined three distinct phenotypes of lungospheres. Ultrastructural and immunohistochemical analyses showed that LSPCs produced more differentiated lung cell progeny. In 3D extracellular matrix, FGF2, FGF7, FGF9, and FGF10 promoted lung organoid formation with similar efficiency. However, FGF9 showed reduced capacity to promote lung organoid formation, suggesting that FGF9 has a reduced ability to sustain LSPCs survival and/or initial divisions. Analysis of lung organoid phenotypes revealed that FGF7 and FGF10 produce bigger organoids and induce organoid branching with higher frequency than FGF2 and FGF9. Higher FGF concentration and/or the use of FGF2 with increased stability and affinity to FGF receptors both increased lung organoid and lungosphere formation efficiency, respectively, suggesting that the level of FGF signaling is a crucial driver of LSPC survival and differentiation, and also lung epithelial morphogenesis. EGF signaling played a supportive but nonessential role in FGF-induced lung organoid formation. Moreover, analysis of tissue architecture and cell type composition confirmed that the lung organoids contained alveolar-like regions with cells expressing alveolar type I and type II cell markers, as well as airway-like structures with club cells and ciliated cells. WNT signaling enhanced the efficiency of lung organoid formation, but in the absence of FGF10 signaling, the organoids displayed limited branching and less differentiated phenotype. In summary, we present lung 3D cell culture models as useful tools to study the role and interplay of signaling pathways in lung development and we reveal roles for FGF ligands in regulation of mouse lung morphogenesisex vivo.</jats:p