Regulation of cell protrusions by small GTPases during fusion of the neural folds.

Epithelial fusion is a crucial process in embryonic development, and its failure underlies several clinically important birth defects. For example, failure of neural fold fusion during neurulation leads to open neural tube defects including spina bifida. Using mouse embryos, we show that cell protrusions emanating from the apposed neural fold tips, at the interface between the neuroepithelium and the surface ectoderm, are required for completion of neural tube closure. By genetically ablating the cytoskeletal regulators Rac1 or Cdc42 in the dorsal neuroepithelium, or in the surface ectoderm, we show that these protrusions originate from surface ectodermal cells and that Rac1 is necessary for the formation of membrane ruffles which typify late closure stages, whereas Cdc42 is required for the predominance of filopodia in early neurulation. This study provides evidence for the essential role and molecular regulation of membrane protrusions prior to fusion of a key organ primordium in mammalian development

Vangl2-environment interaction causes severe neural tube defects, without abnormal neuroepithelial convergent extension

Planar cell polarity (PCP) signalling is vital for initiation of mouse neurulation, with diminished convergent extension (CE) cell movements leading to craniorachischisis, a severe neural tube defect (NTD). Some humans with NTDs also have PCP gene mutations but these are heterozygous, not homozygous as in mice. Other genetic or environmental factors may interact with partial loss of PCP function in human NTDs. We found that reduced sulfation of glycosaminoglycans interacts with heterozygosity for the Lp allele of Vangl2 (a core PCP gene), to cause craniorachischisis in cultured mouse embryos, with rescue by exogenous sulphate. We hypothesised this glycosaminoglycan-PCP interaction may regulate CE but, surprisingly, DiO labeling of the embryonic node demonstrates no abnormality of midline axial extension in sulfation-depleted Lp/+ embryos. Positive-control Lp/Lp embryos show severe CE defects. Abnormalities were detected in the size and shape of somites that flank the closing neural tube in sulfation-depleted Lp/+ embryos. We conclude that failure of closure initiation can arise by a mechanism other than faulty neuroepithelial CE, with possible involvement of matrix-mediated somite expansion, adjacent to the closing neural tube

Mitotane reduces the chemoresistance phenotype in an adrenocortical carcinoma cell line

Adrenocortical carcinoma (ACC), a rare tumor, with incidence of 1–2 per million population annually, has a bimodal distribution by age, with cases clustering in children under 6, and in adults 30–40 years old. ACC has a dismal prognosis. The only curative treatment is complete surgical excision of the tumor, but late diagnosis prevents surgical cure, since ACC frequently recurs and metastasize. Chemotherapy is frequently ineffective, due to the overexpression of the MDR-1 gene, encoding for detoxifying pump P-gp, which confers chemoresistance to ACC. Mitotane, an adrenolitic drug, is being used as adjuvant therapy to prevent recurrence, despite several and important side effects appearing at therapeutic concentrations (14–20 mg/l; 40–60 mM). We aimed at identifying therapeutic strategies to overcome adrenocortical carcinoma chemoresistance by using mitotane. Human adrenocortical cells, NCI-H295R, expressing high P-gp levels, were treated with mitotane (2.5–80 mM) and doxorubicin (1–100 nM), identifying minimal cytotoxic concentrations. Cell viability and caspase 3/7 activity under 25 and 50 nM doxorubicin without or with 5 mM mitotane were tested. These drug concentrations are much lower than those reached in vivo. Combination treatments significantly and more potently reduced cell viability (40–60%) by inducing caspase-3/7 activity. In addition, we found that mitotane significantly reduced P-gp activity, promoting the intracellular accumulation of fluorescent compounds. Our results indicate that mitotane enhances the cytotoxic effects of doxorubicin in a chemoresistant adrenocortical carcinoma cell line, likely inhibiting P-gp activit

Cyclo-oxygenase 2 modulates chemoresistance in breast cancer cells involving NF-kB.

Background: Breast cancer cells can develop chemoresistance after prolonged exposure to cytotoxic drugs due to expression of the multi drug resistance (MDR) 1 gene. Type 2 cyclo-oxygenase (COX-2) inhibitors reverse the chemoresistance phenotype of a medullary thyroid carcinoma cell line, TT, and of a breast cancer cell line, MCF7, by inhibiting MDR1 expression and P-gp function. Aim: investigate the role of prostaglandin (PG) in modulating chemoresistance in MCF7 cells and to explore the involved intracellular mechanisms. Methods: native and chemoresistant MCF7 cells were trated with PGH2 and resistance to Doxorubicin was tested in the presence or absence of COX-2 inhibitors. Results: PGH2 restores resistance to the cytotoxic effects of Doxo, with concomitant nuclear translocation of the transcription factor NF-kB. Conclusions: COX-2 inhibitors prevent chemoresistance development in breast cancer cells by inhibiting P-gp expression and function by a mechanism that involves PGH2 generation and NF-kB activation

mTOR inhibitors hamper cell viability in selected human medullary thyroid carcinoma primary cultures

Introduction: It has been demonstrated that mTOR inhibitors have potent anti-proliferative effects in a human Medullary Thyroid Carcinoma (MTC) cell lines. We here explore the possible role of mTOR inhibitors, Everolimus and BEZ235 (which also inhibits the PI3K pathway) on the effects of Insulin-like Growth Factor-1 (IGF-1) in human MTC primary cultures. Aims: To this purpose, 20 MTCs primary cultures, were treated without or with 1 uM Everolimus, 10 nM BEZ235, and/or 50 nM IGF-1. Materials and methods: Cell viability and apoptosis were evaluated after 48 h. p70S6K phosphorylation was evaluated by ELISA. Results: we observed that Everolimus and BEZ235 significantly reduced MTC cell viability, by 20 and 35% respectively, while IGF-1 enhanced cell viability, an effect completely blocked by mTOR inhibitors. Co-incubation with an IGF-1R blocking antibody enhanced the antiproliferative effects of. Everolimus and BEZ235. Caspase activity was enhanced by BEZ235 and reduced by IGF-1, an effect that was attenuated by co-treatment with mTOR inhibitors. Phosphorylation of p70S6K, a down-stream mTOR effector in the PI3K/Akt pathway, was evaluated to assess whether this effect is due to variations in mTOR activity. We observed that IGF-1 enhanced p70S6K phosphorylation, that is reduced by mTOR inhibitors, indicating that IGF-1 exerts its proliferative effects by inducing this pathway. Conclusion: In conclusion, mTOR inhibitors reduced MTCs cell viability by inducing apoptosis, with a mechanism likely involving IGF-1 signalling, suggesting that these drugs might represent a possible medical treatment for persistent/recurrent MTCs

Magmas overexpression inhibits staurosporine induced apoptosis in rat pituitary adenoma cell lines

Magmas is a nuclear gene that encodes for the mitochondrial import inner membrane translocase subunit Tim16. Magmas is overexpressed in the majority of human pituitary adenomas and in a mouse ACTH-secreting pituitary adenoma cell line. Here we report that Magmas is highly expressed in two out of four rat pituitary adenoma cell lines and its expression levels inversely correlate to the extent of cellular response to staurosporine in terms of apoptosis activation and cell viability. Magmas over-expression in rat GH/PRL-secreting pituitary adenoma GH4C1 cells leads to an increase in cell viability and to a reduction in staurosporine-induced apoptosis and DNA fragmentation, in parallel with the increase in Magmas protein expression. These results indicate that Magmas plays a pivotal role in response to pro-apoptotic stimuli and confirm and extend the finding that Magmas protects pituitary cells from staurosporine-induced apoptosis, suggesting its possible involvement in pituitary adenoma development

PGH2 blocks the chemosensitizing effects of COX-2 inhibitors by a NF-KB mediated mechanism

PGH2 blocks the chemosensitizing effects of COX-2 inhibitors by a NF-KB mediated mechanis

IGF-I proliferative effects are inhibited by targeting PKC in human neuroendocrine tumor cells

IGF-I proliferative effects are inhibited by targeting PKC in human neuroendocrine tumor cell