p21(WAF1 )expression induced by MEK/ERK pathway activation or inhibition correlates with growth arrest, myogenic differentiation and onco-phenotype reversal in rhabdomyosarcoma cells

BACKGROUND: p21(WAF1), implicated in the cell cycle control of both normal and malignant cells, can be induced by p53-dependent and independent mechanisms. In some cells, MEKs/ERKs regulate p21(WAF1 )transcriptionally, while in others they also affect the post-transcriptional processes. In myogenic differentiation, p21(WAF1 )expression is also controlled by the myogenic transcription factor MyoD. We have previously demonstrated that the embryonal rhabdomyosarcoma cell line undergoes growth arrest and myogenic differentiation following treatments with TPA and the MEK inhibitor U0126, which respectively activate and inhibit the ERK pathway. In this paper we attempt to clarify the mechanism of ERK-mediated and ERK-independent growth arrest and myogenic differentiation of embryonal and alveolar rhabdomyosarcoma cell lines, particularly as regards the expression of the cell cycle inhibitor p21(WAF1). RESULTS: p21(WAF1 )expression and growth arrest are induced in both embryonal (RD) and alveolar (RH30) rhabdomyosarcoma cell lines following TPA or MEK/ERK inhibitor (U0126) treatments, whereas myogenic differentiation is induced in RD cells alone. Furthermore, the TPA-mediated post-transcriptional mechanism of p21(WAF1)-enhanced expression in RD cells is due to activation of the MEK/ERK pathway, as shown by transfections with constitutively active MEK1 or MEK2, which induces p21(WAF1 )expression, and with ERK1 and ERK2 siRNA, which prevents p21(WAF1 )expression. By contrast, U0126-mediated p21(WAF1 )expression is controlled transcriptionally by the p38 pathway. Similarly, myogenin and MyoD expression is induced both by U0126 and TPA and is prevented by p38 inhibition. Although MyoD and myogenin depletion by siRNA prevents U0126-mediated p21(WAF1) expression, the over-expression of these two transcription factors is insufficient to induce p21(WAF1). These data suggest that the transcriptional mechanism of p21(WAF1 )expression in RD cells is rescued when MEK/ERK inhibition relieves the functions of myogenic transcription factors. Notably, the forced expression of p21(WAF1 )in RD cells causes growth arrest and the reversion of anchorage-independent growth. CONCLUSION: Our data provide evidence of the key role played by the MEK/ERK pathway in the growth arrest of Rhabdomyosarcoma cells. The results of this study suggest that the targeting of MEK/ERKs to rescue p21(WAF1 )expression and myogenic transcription factor functions leads to the reversal of the Rhabdomyosarcoma phenotype

HDAC inhibition is associated to valproic acid induction of early megakaryocytic markers

Valproic acid (VPA), a histone deacetylase inhibitor, causes differentiation in different cell lines and in a cell-specific manner; yet, its effect on megakaryocytic (MK) differentiation has not been studied. We evaluated whether VPA induces MK differentiation in a UT-7 cell line through histone acetylation in the GpIIIa gene region and activation of the ERK pathway. UT-7 cells, derived from megakaryoblastic leukemia, were treated with VPA at various concentrations, and the expression of differentiation markers as well as the gene expression profile was assessed. Flow cytometry, immunoblot analysis, and RT-PCR demonstrated that VPA induced the expression of the early MK markers GpIIIa (CD61) and GpIIb/IIIa (CD41) in a dose-dependent manner. The VPA-treated cells showed hyperacetylation of the histones H3 and H4; in particular, histone acetylation was found to have been associated with CD61 expression, in that the GpIIIa promoter showed H4 hyperacetylation, as demonstrated by the chromatin immunoprecipitation assay. Furthermore, activation of the ERK pathway was involved in VPA-mediated CD61/CD41 expression and in cell adhesion, as demonstrated by using the MEK/ERK inhibitor U0126. In conclusion, the capacity of VPA to commit UT-7 cells to MK differentiation is mediated by its inhibitory action on HDAC and the long-lived activation of ERK1/2

Key role of MEK/ERK pathway in sustaining tumorigenicity and in vitro radioresistance of embryonal rhabdomyosarcoma stem-like cell population

The identification of signaling pathways that affect the cancer stem-like phenotype may provide insights into therapeutic targets for combating embryonal rhabdomyosarcoma. The aim of this study was to investigate the role of the MEK/ERK pathway in controlling the cancer stem-like phenotype using a model of rhabdospheres derived from the embryonal rhabdomyosarcoma cell line (RD). Rhabdospheres enriched in cancer stem like cells were obtained growing RD cells in non adherent condition in stem cell medium. Stem cell markers were evaluated by FACS analysis and immunoblotting. ERK1/2, myogenic markers, proteins of DNA repair and bone marrow X-linked kinase (BMX) expression were evaluated by immunoblotting analysis. Radiation was delivered using an x-6 MV photon linear accelerator. Xenografts were obtained in NOD/SCID mice by subcutaneously injection of rhabdosphere cells or cells pretreated with U0126 in stem cell medium. MEK/ERK inhibitor U0126 dramatically prevented rhabdosphere formation and down-regulated stem cell markers CD133, CXCR4 and Nanog expression, but enhanced ALDH, MAPK phospho-active p38 and differentiative myogenic markers. By contrast, MAPK p38 inhibition accelerated rhabdosphere formation and enhanced phospho-active ERK1/2 and Nanog expression. RD cells, chronically treated with U0126 and then xeno-transplanted in NOD/SCID mice, delayed tumor development and reduced tumor mass when compared with tumor induced by rhabdosphere cells. U0126 intraperitoneal administration to mice bearing rhabdosphere-derived tumors inhibited tumor growth . The MEK/ERK pathway role in rhabdosphere radiosensitivity was investigated in vitro. Disassembly of rhabdospheres was induced by both radiation or U0126, and further enhanced by combined treatment. In U0126-treated rhabdospheres, the expression of the stem cell markers CD133 and CXCR4 decreased and dropped even more markedly following combined treatment. The expression of BMX, a negative regulator of apoptosis, also decreased following combined treatment, which suggests an increase in radiosensitivity of rhabdosphere cells. Our results indicate that the MEK/ERK pathway plays a prominent role in maintaining the stem-like phenotype of RD cells, their survival and their innate radioresistance. Thus, therapeutic strategies that target cancer stem cells, which are resistant to traditional cancer therapies, may benefit from MEK/ERK inhibition combined with traditional radiotherapy, thereby providing a promising therapy for embryonal rhabdomyosarcoma

Key role of MEK/ERK pathway in sustaining tumorigenicity and in vitro radioresistance of embryonal rhabdomyosarcoma stem-like cell population

The identification of signaling pathways that affect the cancer stem-like phenotype may provide insights into therapeutic targets for combating embryonal rhabdomyosarcoma. The aim of this study was to investigate the role of the MEK/ERK pathway in controlling the cancer stem-like phenotype using a model of rhabdospheres derived from the embryonal rhabdomyosarcoma cell line (RD)

Hypoxia sustains glioblastoma radioresistance through ERKs/DNA-PKcs/HIF-1α functional interplay

The molecular mechanisms by which glioblastoma multiforme (GBM) refracts and becomes resistant to radiotherapy treatment remains largely unknown. This radioresistance is partly due to the presence of hypoxic regions, which are frequently found in GBM tumors. We investigated the radiosensitizing effects of MEK/ERK inhibition on GBM cell lines under hypoxic conditions. Four human GBM cell lines, T98G, U87MG, U138MG and U251MG were treated with the MEK/ERK inhibitor U0126, the HIF-1α inhibitor FM19G11 or γ-irradiation either alone or in combination under hypoxic conditions. Immunoblot analysis of specific proteins was performed in order to define their anti‑oncogenic or radiosensitizing roles in the different experimental conditions. MEK/ERK inhibition by U0126 reverted the transformed phenotype and significantly enhanced the radiosensitivity of T98G, U87MG, U138MG cells but not of the U251MG cell line under hypoxic conditions. U0126 and ERK silencing by siRNA reduced the levels of DNA protein kinase catalytic subunit (DNA-PKcs), Ku70 and K80 proteins and clearly reduced HIF-1α activity and protein expression. Furthermore, DNA-PKcs siRNA-mediated silencing counteracted HIF-1α activity and downregulated protein expression suggesting that ERKs, DNA-PKcs and HIF-1α cooperate in radioprotection of GBM cells. Of note, HIF-1α inhibition under hypoxic conditions drastically radiosensitized all cell lines used. MEK/ERK signal transduction pathway, through the sustained expression of DNA-PKcs, positively regulates HIF-1α protein expression and activity, preserving GBM radioresistance in hypoxic condition

Diagnosis and Treatment of Pulmonary Disease in Sea Turtles (Caretta caretta)

The aim of this study was to describe the clinical signs, radiographic, endoscopic and CT findings, cytological and microbiological findings and treatments of pulmonary diseases in sea turtles, in order to obtain an accurate diagnosis that avoids unnecessary therapy and antibiotic-resistance phenomena. In total, 14 loggerheads (Caretta caretta), with clinical and/or radiographic findings of pulmonary pathology, were assessed through various combinations of clinical, radiological, CT, endoscopic examination and bronchoalveolar lavage, which recovered fluid for cytologic and microbiologic analysis. In all cases, radiographic examination led to a diagnosis of pulmonary disorders—4 unilateral and 10 bilateral. All bacteria cultured were identified as Gram-negative. Antibiotic resistance was greater than 70% for all beta-lactams tested. In addition, all bacterial strains were 100% resistant to colistin sulfate and tetracycline. Specific antibiotic therapies were formulated for seven sea turtles using Enrofloxacin, and for four sea turtles using ceftazidime. In two turtles, antibiotic therapy was not included due to the presence of antibiotic resistance against all the antibiotics evaluated. In both cases, the coupage technique and environmental management allowed the resolution of the lung disease without antibiotics. All 14 sea turtles were released back into the sea. Radiographic examination must be considered the gold standard for screening sea turtles that show respiratory signs or abnormal buoyancy. Susceptibility testing with antimicrobials allowed appropriate therapy, including the reduction of antibiotic-resistance

Correction to: A formulation of neem and hypericum oily extract for the treatment of the wound myiasis by Wohlfahrtia magnifica in domestic animals

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An overview of the Italian forest biodiversity and its conservation level, based on the first outcomes of the 4th Habitat Report ex-Art. 17

In 2019 the 4th Report ex-Art. 17 on the conservation status (CS) of Annex I Habitats of the 92/43/EEC Directive was expected by every EU/28 country, with reference to the period 2013-18. In Italy, the process was in charge to the Italian Institute for Environmental Protection and Research (ISPRA), on behalf of the Ministry for Environment, Land and Sea Protection (MATTM), with the scientific support of the Italian Botanical Society (SBI). A large group of thematic and territorial experts elaborated the available data concerning the 124 types of terrestrial and inland water Habitats present in Italy, 39 of which are represented by Forest Habitats (Group 9),. The main aim of the work was the evaluation of the overall CS of each Habitat by Biogeographic Region (Mediterranean, Continental and Alpine), for a total amount of 294 assessments. A high proportion of these (92, corresponding to 31% of the total) referred to Forest Habitats, including 20 marginal types for which the CS was not requested. The analysis was carried out at different scales: a) administrative territory, through the data contained in the ISPRA database, whose compilation was in charge to the Regions and Autonomous Provinces; b) Natura 2000 site, with the latest updates available (Standard Data Forms updated to 2018); c) national scale, implementing the distribution maps for each Habitat based on the European grid ETRS89-LAEA5210 (10x10 km2 mesh); d) Biogeographic Region, scale of the final assessment. Cartographic outcomes, associated databases and additional data used for the assessments will be available online on the ISPRA Portal as soon as the validation process by the European Commission will be completed. A dedicated archive named "HAB_IT" has been created in the national database "VegItaly" (1), managed by the Italian Society of Vegetation Science, where the phytosociological relevés representative of the various Annex I Habitats in Italy will be archived and freely accessible. An overview of the results regarding the Forest habitats is here provided, including a comparison with the outcomes of the former reporting cycle, the 3rd Report ex-Art. 17 (2). In several cases (e.g. 9120, 91L0), the distribution maps have been remarkably improved due to better knowledge and more fitful interpretation. The conservation status resulted as Favourable (FV) for 6,7%, Inadequate (U1) for 58,7% and Bad (U1) for 32,0% of the 72 assessed forest Habitat types. In no case there was an improvement of the conservation status, while in 6 cases a worsening of the conditions resulted from the data analysis, pointing out the Habitats types with a higher need of action. Similarly to other projects carried out as a team by the network of Annex I Habitat experts of the Italian Botanical Society and the Italian Society for Vegetation Science (e.g. 3, 4), this is another step in the direction of supporting the implementation of the 92/43/EEC "Habitat" Directive in Italy and Europe. On this ground, the high biodiversity of the Italian forest Habitats could be emphasized, however results pointed out that some rare or endemic types (e.g. Alnus cordata or Betula aetnensis-dominated forests) are still scarcely acknowledged by the most prominent EU conservation tools such as the Annex I to the "Habitat" Directive. 1) F. Landucci et al. (2012) Plant Biosyst., 146(4), 756-763 2) P. Genovesi et al. (2014) ISPRA, Serie Rapporti, 194/2014 3) E. Biondi et al. (2009) Società Botanica Italiana, MATTM, D.P.N., http://vnr.unipg.it/habitat/ 4) D. Gigante et al. (2016) Plant Sociology, 53(2), 77-8