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Marine invertebrates represent a valuable reservoir of pharmaceutical bioactive compounds with potential relevance to various medical applications. These compounds exhibit notable advantages when compared to their terrestrial counterparts, in terms of their potency, activity, and mechanism of action. Within this context, the present work aimed to extract, chemically characterize, and investigate the bioactivity of the gonadal extract of the sea urchin Paracentrotus lividus (P. lividus) collected along the Mediterranean coast of Alexandria, Egypt. Fractions of the gonadal extract were characterized by Spectrophotometry and gas chromatography-mass spectrometry (GC-MS), and their bioactivities were investigated in vitro. The analysis supported the extract richness of carotenoids and bioactive compounds. The extract showed promising anticancer activity against three different breast cancer cell lines with different levels of aggressiveness and causative factors, namely MDA-MB-231, MDA-MB-453, and HCC-1954. Gene expression analysis using RT-qPCR showed that P. lividus extract inhibited the expression of crucial factors involved in cell cycle regulation and apoptosis. In addition, the extract significantly inhibited the lipo-polysaccharides (LPS) induced inflammation in the RAW264.7 macrophage cell line and exerted anti-bacterial activity against the Gram-negative bacteria Klebsiella pneumoniae and Pseudomonas aeruginosa. Collectively, these results demonstrated the chemical richness and the wide-scale applicability of P. lividus gonadal extract as an anti-cancer, anti-bacterial, and anti-inflammatory natural extract.

The present study investigates the neuroprotective effects of the sea urchin Paracentrotus lividus gonadal extract on rotenone-induced neurotoxicity in a Parkinson's disease (PD) rat model. Parkinson's disease, characterized by the progressive loss of dopaminergic neurons in the substantia nigra (SN), is exacerbated by oxidative stress and neuroinflammation. The study involved fifty Wistar rats divided into five groups: control, dimethyl sulfoxide (DMSO) control, Paracentrotus lividus gonadal extract-treated, rotenone-treated, and combined rotenone with Paracentrotus lividus gonadal extract-treated. Behavioral assessments included the rotarod and open field tests, while biochemical analyses measured oxidative stress markers (malondialdehyde (MDA), nitric oxide (NO), glutathione (GSH)), antioxidants (superoxide dismutase (SOD), catalase (CAT)), pro-inflammatory cytokines (interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha)), and neurotransmitters (dopamine (DA), levodopa (L-Dopa)). Histological and immunohistochemical analyses evaluated the neuronal integrity and tyrosine hydroxylase (TH) and alpha-synuclein expression. The results showed that Paracentrotus lividus gonadal extract significantly mitigated rotenone-induced motor deficits and improved locomotor activity. Biochemically, the extract reduced oxidative stress and inflammation markers while enhancing antioxidant levels. Histologically, it restored neuronal integrity and reduced alpha-synuclein accumulation. Molecularly, it increased tyrosine hydroxylase and dopa decarboxylase gene expression, essential for dopamine synthesis. These findings suggest that Paracentrotus lividus gonadal extract exerts neuroprotective effects by modulating oxidative stress, neuroinflammation, and dopaminergic neuron integrity, highlighting its potential as a therapeutic agent for Parkinson's disease.

Background: p63 is a transcription factor with intrinsic pioneer factor activity and pleiotropic functions. Transforming growth factor beta (TGF beta) signaling via activation and cooperative action of canonical, SMAD, and non-canonical, MAP-kinase (MAPK) pathways, elicits both anti- and pro-tumorigenic properties, including cell stemness and invasiveness. TGF beta activates the Delta Np63 transcriptional program in cancer cells; however, the link between TGF beta and p63 in unmasking the epigenetic landscape during tumor progression allowing chromatin accessibility and gene transcription, is not yet reported.

Methods: Small molecule inhibitors, including protein kinase inhibitors and RNA-silencing, provided loss of function analyses. Sphere formation assays in cancer cells, chromatin immunoprecipitation and mRNA expression assays were utilized in order to gain mechanistic evidence. Mass spectrometry analysis coupled to co-immunoprecipitation assays revealed novel p63 interactors and their involvement in p63-dependent transcription.

Results: The sphere-forming capacity of breast cancer cells was enhanced upon TGF beta stimulation and significantly decreased upon Delta Np63 depletion. Activation of TGF beta signaling via p38 MAPK signaling induced Delta Np63 phosphorylation at Ser 66/68 resulting in stabilized Delta Np63 protein with enhanced DNA binding properties. TGF beta stimulation altered the ratio of H3K27ac and H3K27me3 histone modification marks, pointing towards higher H3K27ac and increased p300 acetyltransferase recruitment to chromatin. By silencing the expression of Delta Np63, the TGF beta effect on chromatin remodeling was abrogated. Inhibition of H3K27me3, revealed the important role of TGF beta as the upstream signal for guiding Delta Np63 to the TGF beta/SMAD gene loci, as well as the indispensable role of Delta Np63 in recruiting histone modifying enzymes, such as p300, to these genomic regions, regulating chromatin accessibility and gene transcription. Mechanistically, TGF beta through SMAD activation induced dissociation of Delta Np63 from NURD or NCOR/SMRT histone deacetylation complexes, while promoted the assembly of Delta Np63-p300 complexes, affecting the levels of histone acetylation and the outcome of Delta Np63-dependent transcription.

Conclusions: Delta Np63, phosphorylated and recruited by TGF beta to the TGF beta/SMAD/Delta Np63 gene loci, promotes chromatin accessibility and transcription of target genes related to stemness and cell invasion.

Cutaneous squamous cell carcinoma (cSCC) is a fast-increasing cancer with metastatic potential. Extracellular vesicles (EVs) are small membrane-bound vesicles that play important roles in intercellular communication, particularly in the tumor microenvironment (TME). Here we report that cSCC cells secrete an increased number of EVs relative to normal human epidermal keratinocytes (NHEKs) and that interfering with the capacity of cSCC to secrete EVs inhibits tumor growth in vivo in a xenograft model of human cSCC. Transcriptome analysis of tumor xenografts by RNA-sequencing enabling the simultaneous quantification of both the human and the mouse transcripts revealed that impaired EV-production of cSCC cells prominently altered the phenotype of stromal cells, in particular genes related to extracellular matrix (ECM)-formation and epithelial-mesenchymal transition (EMT). In line with these results, co-culturing of human dermal fibroblasts (HDFs) with cSCC cells, but not with normal keratinocytes in vitro resulted in acquisition of cancer-associated fibroblast (CAF) phenotype. Interestingly, EVs derived from metastatic cSCC cells, but not primary cSCCs or NHEKs, were efficient in converting HDFs to CAFs. Multiplex bead-based flow cytometry assay and mass-spectrometry (MS)-based proteomic analyses revealed the heterogenous cargo of cSCC-derived EVs and that especially EVs derived from metastatic cSCCs carry proteins associated with EV-biogenesis, EMT, and cell migration. Mechanistically, EVs from metastatic cSCC cells result in the activation of TGFβ signaling in HDFs. Altogether, our study suggests that cSCC-derived EVs mediate cancer-stroma communication, in particular the conversion of fibroblasts to CAFs, which eventually contribute to cSCC progression.

One of the central regulators of cell growth, proliferation, and metabolism is the mammalian target of rapamycin, mTOR, which exists in two structurally and functionally different complexes: mTORC1 and mTORC2; unlike m TORC2, mTORC1 is activated in response to the sufficiency of nutrients and is inhibited by rapamycin. mTOR complexes have critical roles not only in protein synthesis, gene transcription regulation, proliferation, tumor metabolism, but also in the regulation of the programmed cell death mechanisms such as autophagy and apoptosis. Autophagy is a conserved catabolic mechanism in which damaged molecules are recycled in response to nutrient starvation. Emerging evidence indicates that the mTOR signaling pathway is frequently activated in tumors. In addition, dysregulation of autophagy was associated with the development of a variety of human diseases, such as cancer and aging. Since mTOR can inhibit the induction of the autophagic process from the early stages of autophagosome formation to the late stage of lysosome degradation, the use of mTOR inhibitors to regulate autophagy could be considered a potential therapeutic option. The present review sheds light on the mTOR and autophagy signaling pathways and the mechanisms of regulation of mTOR-autophagy.

Simple Summary The hyaluronan receptor CD44 has an important role in glioblastoma multiforme (GBM) progression, but the precise mechanisms have not been elucidated. We have analyzed U251MG glioma cells, expressing CD44 or not, and grown in stem cell-like enriched spheres. Our results revealed that CD44 is important for cell growth and stemness, and for the prevention of senescence. Analysis by RNA sequencing revealed that CD44 is important for the interaction with the hyaluronan-enriched microenvironment. In addition, CD44 depletion impairs certain gene signatures, such as those for platelet-derived growth factor (PDGF) isoforms and PDGF receptors, as well as signatures related to hypoxia, glycolysis, and anti-tumor immune responses. Glioblastoma multiforme (GBM) is a lethal brain tumor, characterized by enhanced proliferation and invasion, as well as increased vascularization and chemoresistance. The expression of the hyaluronan receptor CD44 has been shown to correlate with GBM progression and poor prognosis. Here, we sought to elucidate the molecular mechanisms by which CD44 promotes GBM progression by knocking out (KO) CD44, employing CRISPR/Cas9 gene editing in U251MG cells. CD44-depleted cells exhibited an impaired proliferation rate, as shown by the decreased cell numbers, decreased Ki67-positive cell nuclei, diminished phosphorylation of CREB, and increased levels of the cell cycle inhibitor p16 compared to control cells. Furthermore, the CD44 KO cells showed decreased stemness and increased senescence, which was manifested upon serum deprivation. In stem cell-like enriched spheres, RNA-sequencing analysis of U251MG cells revealed a CD44 dependence for gene signatures related to hypoxia, the glycolytic pathway, and G2 to M phase transition. Partially similar results were obtained when cells were treated with the gamma-secretase inhibitor DAPT, which inhibits CD44 cleavage and therefore inhibits the release of the intracellular domain (ICD) of CD44, suggesting that certain transcriptional responses are dependent on CD44-ICD. Interestingly, the expression of molecules involved in hyaluronan synthesis, degradation, and interacting matrix proteins, as well as of platelet-derived growth factor (PDGF) isoforms and PDGF receptors, were also deregulated in CD44 KO cells. These results were confirmed by the knockdown of CD44 in another GBM cell line, U2990. Notably, downregulation of hyaluronan synthase 2 (HAS2) impaired the hypoxia-related genes and decreased the CD44 protein levels, suggesting a CD44/hyaluronan feedback circuit contributing to GBM progression.

The transcription factor SNAI1 mediates epithelial-mesenchymal transition, fibroblast activation and controls inter-tissue migration. High SNAI1 expression characterizes metastatic triple-negative breast carcinomas, and its knockout by CRISPR/Cas9 uncovered an epithelio-mesenchymal phenotype accompanied by reduced signaling by the cytokine TGFβ. The SNAI1 knockout cells exhibited plasticity in differentiation, drifting towards the luminal phenotype, gained stemness potential and could differentiate into acinar mammospheres in 3D culture. Loss of SNAI1 de-repressed the transcription factor FOXA1, a pioneering factor of mammary luminal progenitors. FOXA1 induced a specific gene program, including the androgen receptor (AR). Inhibiting AR via a specific antagonist regenerated the basal phenotype and blocked acinar differentiation. Thus, loss of SNAI1 in the context of triple-negative breast carcinoma cells promotes an intermediary luminal progenitor phenotype that gains differentiation plasticity based on the dual transcriptional action of FOXA1 and AR. This function of SNAI1 provides means to separate cell invasiveness from progenitor cell de-differentiation as independent cellular programs.