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Rhabdomyosarcoma (RMS) is a highly malignant and metastatic pediatric cancer that arises from the skeletal muscle. Recent studies have identified an important role of AKT signaling in RMS progression. This suggests targeting components of the PI3K/Akt pathway may be an effective therapeutic strategy. Here, we investigated the in vitro activity of the class I PI3K inhibitors [1] in human rhabdomyosarcoma cell lines (embryonal rhabdomyosarcoma RD and A204, alveolar rhabdomyosarcoma SJCRH30). We used a panel of four compounds which specifically target PI3K isoforms including the PI3Kα-selective (p110α ) inhibitor alpelisib BYL719, currently in clinical development by Novartis Oncology, the p110β TGX-221 inhibitor, the p110γ CZC24832, the p110δ CAL-101inhibitor and the dual p110α/p110δ inhibitor AZD8835. The effects of single drugs and of several drug combinations were analyzed to assess cytotoxicity by MTT assays, cell cycle by flow cytometry , apoptosis by caspase 3/7 assay and Western blot, as well as the phosphorylation status of the pathway. BYL719 treatment resulted in G1 phase cell cycle arrest and apoptosis. BYL719 administered in combination with CAL-101, for 48 h and 72h, decreased cell viability and induced apoptosis in a marked synergistic manner. Taken together, our findings indicate that BYL719, either alone or in combination with p110δ CAL-101inhibitor, may be an efficient treatment for human rhabdomyosarcoma cells that have aberrant upregulation of the PI3K signaling pathway for their proliferation and survival., Italian Journal of Anatomy and Embryology, Vol. 122, No. 1 (Supplement) 2017

Prohibitins (PHBs) are a highly conserved class of proteins first discovered as inhibitors of cellular proliferation. Since then PHBs have been found to have a significant role in transcription, nuclear signaling, mitochondrial structural integrity, cell division, and cellular membrane metabolism, placing these proteins among the key regulators of pathologies such as cancer, neuromuscular degeneration, and other metabolic diseases. The human genome encodes two PHB proteins, prohibitin 1 (PHB1) and prohibitin 2 (PHB2), which function not only as a heterodimeric complex, but also independently. While many previous reviews have focused on the better characterized prohibitin, PHB1, this review focuses on PHB2 and new data concerning its cellular functions both in complex with PHB1 and independent of PHB1. © 2015 IUBMB Life, 67(4):239–254, 2015

PLC-beta 1 (PLCβ1) inhibits erythroid differentiation induced by mithramycin (MTH) by targeting miR-210 expression. MicroRNA-210 (miR-210) has been reported to be upregulated in various types of human malignancy suggesting that it has an important role in tumorigenesis. Inhibition of miR-210 affects the erythroid differentiation pathway and it occurs to a greater extent in MTH-treated cells. In this paper we have analyzed the effect of MTH on human K562 cells differentiation. Overexpression of PLCβ1 suppresses the differentiation of K562 elicited by MTH as demonstrated by the absence of γ-globin expression. Inhibition of PLCβ1 expression is capable to promote the differentiation process leading to a recovery of γ-globin gene even in the absence of MTH. Our experimental evidences suggest that PLCβ1 signalling regulates erythropoiesis through miR-210. Indeed overexpression of PLCβ1 leads to a decrease of miR-210 expression after MTH treatment. Moreover miR-210 is up-regulated through both proliferation and differentiation events when PLCβ1 expression is down-regulated. Therefore we suggest a novel role for PLCβ1 in regulating miR-210 and our data hint at the fact that, in human K562 erythroleukemia cells, the modulation of PLCβ1 expression is able to exert an impairment of normal erythropoiesis as assessed by γ-globin expression., Italian Journal of Anatomy and Embryology, Vol 119, No 1 (Supplement) 2014

The double-strand RNA-dependent protein kinase, PKR, plays a central role in inflammatory/chronic stress-mediated pathologies such as cancer, diabetes, and neuro/muscular degenerative diseases. Although a significant amount of research has been conducted to elucidate the role of PKR signaling in the cytosol, only recently has attention been paid to the role of PKR in the nuclear compartment. Previously our group reported that phosphorylated forms of PKR are present in the nucleus of acute leukemic cell lines, representing a reservoir of active kinase that responds to stress. Using the CCRF-CEM acute T-cell leukemia cell line, a PKR-specific inhibitor, co-immunoprecipitation and a proteomics approach, which included affinity purified mass spectrometry analysis (AP/MS), we identified the proteins present in active and inactive PKR nuclear complexes. Of the proteins identified in the PKR complexes, sixty-nine (69) were specific to the active complex, while thirty-eight (38) were specific to the inactive complex. An additional thirteen (13) proteins associated specifically with both complexes. The majority of the proteins identified are involved in, ribosome biogenesis, RNA splicing, mRNA stability, gene expression, cell cycle, or chromatin organization, including several with known significance to normal hematopoiesis and/or hematological disease. In agreement with the AP/MS data, basal- or over-expression of PKR under normal growth conditions favored cell proliferation in the tested cell lines, whereas pharmacological inhibition of PKR or shRNA-mediated knock-down did not. PKR was also found to influence the isoform and the level of expression of the proto-oncogene MYC.

Inositide-dependent phospholipase Cβ1 (PI-PLCβ1b) has two isoforms generated by alternative splicing (PI-PLCβ1a and PI-PLCβ1b). In murine erythroleukemia (MEL) cells both the isoforms are present within the nucleus, but PI-PLCβ1b is exclusively nuclear. Our group has demonstrated that PI-PLCβ1 nuclear localisation is crucial for its function, although the mechanism by which PI-PLCβ1 is imported into the nucleus has never been carefully investigated. The purpose of the present study was to get more insights on the protein interactome of PI-PLCβ1b, namely the proteins present in the nucleus. Immuno-affinity purification coupled with tandem mass spectrometry analysis have been used to purify and identify PI-PLCβ1b interaction binding partners from Friend’s erythroleukemia isolated nuclei. Gene ontology and protein-protein interaction network were performed to analyze data. Some interactions were already characterized, such as the binding with the splicing factor SRp20 and the lamin B. Among the proteins identified, the binding of eEF1A and prohibitin 2 with PI-PLCβ1b was confirmed by western blot analysis. Of particular interest was the identification of importin a, importin b1 and Ran, which interact with PI-PLCβ1b. These proteins are believed to be involved in the import mechanism from the cytoplasm to the nucleus. Further analysis by overexpressing both wild type and cytoplasmatic mutant of PI-PLCβ1, suggests that importin b1 is responsible for the localisation of PI-PLCβ1b in the nucleus, giving new insight into the mechanism of trafficking of this signaling molecule., Italian Journal of Anatomy and Embryology, Vol 117, No 2 (Supplement) 2012

PURPOSE: To evaluate the tear protein pattern in patients with recent subjective symptoms of dry eye (DE) and with poor distinctive DE clinical signs. METHODS: One hundred sixty patients suspected of suffering from mild to moderate DE according to the Dry Eye Workshop (DEWS report 2007) severity grade and 45 matched normal volunteers were included in the study. Subjective symptom score (Ocular Surface Disease index score), Schirmer test I, tear film break-up time, cornea and conjunctiva staining (National Eye Institute score); and tear protein analysis were performed. Statistical evaluation of data was performed with Mann-Whitney unpaired and Student t tests, (significance p

Italian Journal of Anatomy and Embryology, Vol 115, No 1/2 (Supplement) 2010