Cristiana Griffoni

MiR-101 downregulation is involved in cyclooxygenase-2 overexpression in human colon cancer cells.

Overexpressed cyclooxygenase-2 (COX-2) strongly contributes to the growth and invasiveness of tumoral cells in patients affected by colorectal cancer (CRC). It has been demonstrated that COX-2 overexpression depends on different cellular pathways involving both transcriptional and post-transcriptional regulations. We assumed that COX-2 expression could be regulated also by microRNAs (miRNAs) since these short RNA molecules participate to the fine regulation of several genes implicated in cell growth and differentiation. In this paper, we report the inverse correlation between COX-2 and miR-101 expression in colon cancer cell lines and we demonstrated in vitro the direct inhibition of COX-2 mRNA translation mediated by miR-101. Moreover, this correlation was supported by data collected ex vivo, in which colon cancer tissues and liver metastases derived from CRC patients were analyzed. These findings provide a novel molecular insight in the modulation of COX-2 at post-transcriptional level by miR-101 and strengthen the observation that miRNAs are highly implicated in the control of gene expression. An impairment of miR-101 levels could represent one of the leading causes of COX-2 overexpression in colon cancer cells.

Involvement of caveolae and caveolae-like domains in signalling, cell survival and angiogenesis

Caveolae, the flask-shaped membrane invaginations abundant in endothelial cells, have acquired a prominent role in signal transduction. Evidence, that events occurring in caveolae participate in cell survival and angiogenesis, has been recently substantiated by the identification of two novel caveolar constituents: prostacyclin synthase (PGIS) and the cellular form of prion protein (PrP(c)). We have shown that PGIS, previously described as an endoplasmic reticulum component, is bound to caveolin-1 (cav-1) and localized in caveolae in human endothelial cells. By generating prostacyclin, PGIS is involved in angiogenesis. Previous observations regarding the localization of PrP(c) in caveolae-like membrane domains (CLDs) have been recently confirmed and extended. It has been demonstrated that PrP(c) is bound to cav-1 and, by recruiting Fyn kinase, can participate in signal transduction events connected to cell survival and differentiation. The new entries of PGIS and PrP(c) in caveolar components place caveolae and CLDs at the centre of a network, where cells decide whether to proliferate or differentiate and whether to survive or to suicide by apoptosis.

Mechanosensing Role of Caveolae and Caveolar Constituents in Human Endothelial Cells

A variety of evidence suggests that endothelial cell functions are impaired in altered gravity conditions. Nevertheless, the effects of hypergravity on endothelial cell physiology remain unclear. In this study we cultured primary human endothelial cells under mild hypergravity conditions for 24–48 h, then we evaluated the changes in cell cycle progression, caveolin1 gene expression and in the caveolae status by confocal microscopy. Moreover, we analyzed the activity of enzymes known to be resident in caveolae such as endothelial nitric oxide synthase (eNOS), cycloxygenase 2 (COX‐2), and prostacyclin synthase (PGIS). Finally, we performed a three‐dimensional in vitro collagen gel test to evaluate the modification of the angiogenic responses. Results indicate that hypergravity shifts endothelial cells to G0/G1 phase of cell cycle, reducing S phase, increasing caveolin1 gene expression and causing an increased distribution of caveolae in the cell interior. Hypergravity also increases COX‐2 expression, nitric oxide (NO) and prostacyclin (PGI2) production, and inhibits angiogenesis as evaluated by 3‐D collagen gel test, through a pathway not involving apoptosis. Thus, endothelial cell caveolae may be responsible for adaptation of endothelium to hypergravity and the mechanism of adaptation involves an increased caveolin1 gene expression coupled to upregulation of vasodilators as NO and PGI2. J. Cell. Physiol. 197: 198–204, 2003.

Redundancy of autocrine loops in human rhabdomyosarcoma cells: induction of differentiation by suramin.

Three human rhabdomyosarcoma cell lines were used to investigate the presence of autocrine loops based on the production of insulin-like growth factor (IGF)-II, basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF)/transforming growth factor (TGF)-alpha and of their corresponding receptors, and whether these loops affect cell proliferation and myogenic differentiation. Two cell lines, RD/18 and CCA, deriving from tumours of the embryonal histotype, showed the presence of both growth factors and receptors which make possible three different autocrine loops, while the alveolar RMZ-RC2 cell line lacked that based on the EGF receptor. Culture of rhabdomyosarcoma cells in the presence of specific blocking antibodies, directed to a component of single autocrine loops, inhibited cell proliferation (up to 50%), without inducing myogenic differentiation. Suramin, a drug which non-selectively interferes with the binding of growth factors to their cellular receptors, was used to block all the autocrine loops simultaneously. In CCA and RMZ-RC2 cells suramin was able to induce a significant increase (up to 3-fold) in the proportion of myosin-positive cells over control cultures. Therefore rhabdomyosarcoma cells of embryonal and alveolar histotype can show a redundancy of growth-sustaining autocrine loops. Suramin could interfere with them by acting on both growth inhibition and induction of myogenic differentiation.

Cellular prion protein and caveolin-1 interaction in a neuronal cell line precedes Fyn/Erk 1/2 signal transduction.

It has been reported that cellular prion protein (PrPc) is enriched in caveolae or caveolae-like domains with caveolin-1 (Cav-1) participating to signal transduction events by Fyn kinase recruitment. By using the Glutathione-S-transferase (GST)-fusion proteins assay, we observed that PrPc strongly interacts in vitro with Cav-1. Thus, we ascertained the PrPc caveolar localization in a hypothalamic neuronal cell line (GN11), by confocal microscopy analysis, flotation on density gradient, and coimmunoprecipitation experiments. Following the anti-PrPc antibody-mediated stimulation of live GN11 cells, we observed that PrPc clustered on plasma membrane domains rich in Cav-1 in which Fyn kinase converged to be activated. After these events, a signaling cascade through p42/44 MAP kinase (Erk 1/2) was triggered, suggesting that following translocations from rafts to caveolae or caveolaelike domains PrPc could interact with Cav-1 and induce signal transduction events.

Modification of proteins secreted by endothelial cells during modeled low gravity exposure

The exposure of the human body to microgravity, conditions that occurs during space flights, causes significant changes in the cardiovascular system. Many cell types have been involved in these changes, and the endothelium seems to play a major role. In endothelial cells (EC), it has been shown that modeled low gravity impairs nitric oxide synthesis, cell adhesion, extracellular matrix composition, cytoskeleton organization, cytokines, and growth factors secretion. Nevertheless, detailed analysis of EC physiological changes induced by microgravity exposure is still lacking. Secretome analysis is one of the most promising approaches for the identification of biomarkers directly related to the physiopathological cellular state. In this study, we analyzed in details the modifications of EC secretome by using umbilical vein endothelial (HUVE) cells exposed to modeled low gravity conditions. By adopting a two‐dimensional (2‐D) proteomic approach, in conjunction with a technique for the compression of the dynamic range of proteins, we observed that modeled low gravity exposure of HUVE cells affected the secretion of proteins involved in the regulation of cytoskeleton assembly. Moreover, by using Luminex® suspension array systems, we found that the low gravity condition decreased in ECs the secretion of some key pro‐inflammatory cytokines, including IL‐1α and IL‐8, and of the pro‐angiogenic factor bFGF. On the contrary, microgravity increase the secretion of two chemokines (Rantes and Eotaxin), involved in leukocytes recruitment. J. Cell. Biochem. 112: 265–272, 2011.

RNAi-Based Strategies for Cyclooxygenase-2 Inhibition in Cancer

Cyclooxygenase-2 (COX-2) enzyme has been involved in the tumorigenesis and in the progression of colorectal cancer (CRC). The use of traditional nonsteroidal anti-inflammatory drugs (NSAIDs) or selective COX-2 inhibitors has been proposed for the prevention and the treatment of this relevant neoplastic disease. In the light of an innovative alternative to these pharmacological approaches, we review here the possible strategies to achieve a strong and selective inhibition of COX-2 enzyme by using the mechanism of RNA Interference (RNAi) targeted against its mRNA. Anti-COX-2 siRNA molecules (siCOX-2) can be generated in CRC cells from short hairpin RNA (shRNA) precursors, delivered in vitro by a retroviral expression system, and induce a significant and stable silencing of overexpressed COX-2 in human colon cancer cells. As a safer alternative to viral approach, nonpathogenic bacteria (E. coli) can be engineered to invade eukaryotic cells and to generate siCOX-2 molecules in cancer cells. Moreover, the involvement of miRNAs in COX-2 posttranscriptional regulation opens up the possibility to exploit an endogenous silencing mechanism to knockdown overexpressed COX-2. Thus, these recent strategies disclose new challenging perspectives for the development of clinically compatible siRNA or miRNA capable of selectively inhibiting COX-2 enzyme.

RNA interference as a key to knockdown overexpressed cyclooxygenase-2 gene in tumour cells

Silencing those genes that are overexpressed in cancer and contribute to the survival and progression of tumour cells is the aim of several researches. Cyclooxygenase-2 (COX-2) is one of the most intensively studied genes since it is overexpressed in most tumours, mainly in colon cancer. The use of specific COX-2 inhibitors to treat colon cancer has generated great enthusiasm. Yet, the side effects of some inhibitors emerging during long-term treatment have caused much concern. Genes silencing by RNA interference (RNAi) has led to new directions in the field of experimental oncology. In this study, we detected sequences directed against COX-2 mRNA, that potently downregulate COX-2 gene expression and inhibit phorbol 12-myristate 13-acetate-induced angiogenesis in vitro in a specific, nontoxic manner. Moreover, we found that the insertion of a specific cassette carrying anti-COX-2 short hairpin RNA sequence into a viral vector (pSUPER.retro) greatly increased silencing potency in a colon cancer cell line (HT29) without activating any interferon response. Phenotypically, COX-2 deficient HT29 cells showed a significant impairment of their in vitro malignant behaviour. Thus, the retroviral approach enhancing COX-2 knockdown, mediated by RNAi, proved to be an useful tool to better understand the role of COX-2 in colon cancer. Furthermore, the higher infection efficiency we observed in tumour cells, if compared to normal endothelial cells, may disclose the possibility to specifically treat tumour cells without impairing endothelial COX-2 activity.

Extracellular copper ions regulate cellular prion protein (PrPC) expression and metabolism in neuronal cells

The physiological functions of cellular prion protein (PrPC) remain unclear. It has been demonstrated that PrPC is a copper binding protein and proposed that its functions could be strictly linked to copper metabolism and neuroprotection. The aim of this study was to clarify how extracellular copper modifies PrPC expression and metabolism in cultured neurones. We reported here that copper delivered at physiological concentrations significantly decreases PrPC mRNA expression in GN11 neurones. Moreover, copper increases the release of PrPC into the culture medium. These results indicate that extracellular copper strongly affects the amount of cellular PrP and might represent an interesting strategy to decrease the expression of PrPC in neurones and its conversion in the pathological isoform PrPSc.

Expression of interleukin 15 (IL-15) in human rhabdomyosarcoma, osteosarcoma and Ewing's sarcoma.

Interleukin 15 (IL‐15) is a recently discovered cytokine that stimulates lymphocyte proliferation and migration via a trimeric receptor sharing the β and γ signal transducing chains with the IL‐2 receptor. IL‐15 is typically produced by normal cells that do not release IL‐2, but little information is currently available on human tumors. To assess whether human musculo‐skeletal sarcomas produce IL‐15, we analyzed surgical specimens and cell lines obtained from rhabdomyosarcoma, osteosarcoma and Ewings sarcoma. IL‐15 mRNA was present in 9/9 surgical specimens (3 Ewings sarcomas, 5 osteosarcomas and 1 rhabdomyosarcoma). The analysis of a panel of cell lines (7 derived from Ewings sarcoma, 12 from osteosarcoma and 5 from rhabdomyosarcoma) showed that all rhabdomyosarcoma and osteosarcoma cell lines expressed IL‐15 mRNA at levels ranging from low to high, while Ewings sarcoma cells contained little or no IL‐15 message. ELISA assays showed IL‐15 release in a subset of rhabdomyosarcomas and osteosarcomas, but not in Ewings sarcoma. The highest production of IL‐15, in the picogram/ml range, was found in rhabdomyosarcoma cell lines RH30 and RD. Int. J.Cancer 71:732‐736, 1997.