Sphingosine Kinase Mediates Resistance to the Synthetic Retinoid N-(4-Hydroxyphenyl)retinamide in Human Ovarian Cancer Cells

A2780 human ovarian carcinoma cells respond to treatment with the synthetic retinoid N-(4-hydroxyphenyl)retinamide (HPR) with the production of dihydroceramide and with a concomitant reduction of cell proliferation and induction of apoptosis. The derived HPR-resistant clonal cell line, A2780/HPR, is less responsive to HPR in terms of dihydroceramide generation. In this report, we show that the production of sphingosine 1-phosphate (S1P) is significantly higher in A2780/HPR versus A2780 cells due to an increased sphingosine kinase (SK) activity and SK-1 mRNA and protein levels. Treatment of A2780 and A2780/HPR cells with a potent and highly selective pharmacological SK inhibitor effectively reduced S1P production and resulted in a marked reduction of cell proliferation. Moreover, A2780/HPR cells treated with a SK inhibitor were sensitized to the cytotoxic effect of HPR, due to an increased dihydroceramide production. On the other hand, the ectopic expression of SK-1 in A2780 cells was sufficient to induce HPR resistance in these cells. Challenge of A2780 and A2780/HPR cells with agonists and antagonists of S1P receptors had no effects on their sensitivity to the drug, suggesting that the role of SK in HPR resistance in these cells is not mediated by the S1P receptors.These data clearly demonstrate a role for SK in determining resistance to HPR in ovarian carcinoma cells, due to its effect in the regulation of intracellular ceramide/S1P ratio, which is critical in the control of cell death and proliferation.     

The co-chaperone BAG3 interacts with the cytosolic chaperonin CCT: New hints for actin folding

It has been recently hypothesized that BAG3 protein, a co-chaperone of Hsp70/Hsc70, is involved in the regulation of several cell processes, such as apoptosis, autophagy and cell motility. Following the identification of Hsc70/Hsp70, further BAG3 molecular partners such as PLC-γ and HspB8 were likewise identified, thus contributing to the characterization of the mechanisms and the biological roles carried out by this versatile protein. By using a His-tagged BAG3 protein as bait, we fished out and identified the cytosolic chaperonin CCT, a new unreported BAG3 partner. The interaction between BAG3 and CCT was confirmed and characterized by co-immunoprecipitation experiments and surface plasmon resonance techniques. Furthermore, our analyses showed a slower CCT association and a faster dissociation with a truncated form of BAG3 containing the BAG domain, thus indicating that other protein regions are essential for a high-affinity interaction. ATP or ADP does not seem to significantly influence the chaperonin binding to BAG3 protein. On the other hand, our experiments showed that BAG3 silencing by small interfering RNA slowed down cell migration and influence the availability of correctly folded monomeric actin, analyzed by DNAse I binding assays and latrunculin A depolymerization studies. To our knowledge, this is the first report showing a biologically relevant interaction between the chaperonin CCT and BAG3 protein, thus suggesting interesting involvement in the folding processes regulated by CCT.     

Misregulated E-cadherin expression associated with an aggressive brain tumor phenotype

Background

Cadherins are essential components of the adherens junction complexes that mediate cell-cell adhesion and regulate cell motility. During tissue morphogenesis, changes in cadherin expression (known as cadherin switching) are a common mechanism for altering cell fate. Cadherin switching is also common during epithelial tumor progression, where it is thought to promote tumor invasion and metastasis. E-cadherin is the predominant cadherin expressed in epithelial tissues, but its expression is very limited in normal brain.

Methodology/Principal Findings

We identified E-cadherin expression in a retrospective series of glioblastomas exhibiting epithelial or pseudoepithelial differentiation. Unlike in epithelial tissues, E-cadherin expression in gliomas correlated with an unfavorable clinical outcome. Western blotting of two panels of human GBM cell lines propagated either as xenografts in nude mice or grown under conventional cell culture conditions confirmed that E-cadherin expression is rare. However, a small number of xenograft lines did express E-cadherin, its expression correlating with increased invasiveness when the cells were implanted orthotopically in mouse brain. In the conventionally cultured SF767 glioma cell line, E-cadherin expression was localized throughout the plasma membrane rather than being restricted to areas of cell-cell contact. ShRNA knockdown of E-cadherin in these cells resulted in decreased proliferation and migration in vitro.

Conclusions/Significance

Our data shows an unexpected correlation between the abnormal expression of E-cadherin in a subset of GBM tumor cells and the growth and migration of this aggressive brain tumor subtype.     

AQP1 is not only a water channel: It contributes to cell migration through Lin7/β-catenin

AQPs are water channel proteins. In particular, AQP1 was demonstrated to be involved in cell migration. According to the model proposed by Verkman and collaborators, AQP drives water influx, facilitating lamellipodia extension and cell migration. Investigating the possible connection between AQP1 and cytoskeleton, our group showed that such a water channel through Lin7/β-catenin affects the organization of the cytoskeleton and proposed a model.All together, these data appear particularly intriguing since the use of AQP1 as target might be useful to modulate angiogenesis/vasculogenic mimicry.Key words: AQP, cytoskeleton, Lin7, β-catenin, motility, molecular adhesionAquaporins were discovered by Peter Agre, who won the Nobel Prize in Chemistry in 2003. They are a family of water-specific, membrane-channel proteins expressed in diverse tissues. Two functional groups of mammalian aquaporins are now recognized: aquaporins (AQP1, AQP2, AQP4, AQP5 and AQP8) which are primarily water selective and aquaglyceroporins (AQP3, AQP7, AQP9 and AQP10) which are permeable to small uncharged solutes such as lactate, glycerol and urea in addition to water.¹ The characterization of the organization of aquaporin genes and identification of their position within the human and mouse genomes have established a primary role for some aquaporins in clinical disorders such as congenital cataracts and nephrogenic diabetes insipidus.² More recently, in the control of fat accumulation, aquaporins were demonstrated to play an important role.^3–6 A characterization of AQPs was recently carried out in neuronal stem cells.⁷ More interesting, an impairment of endothelial cell migration, without altering their proliferation or adhesion, was shown by AQP1 null mice.⁸ Based on findings of slowed lamellipodial dynamics in AQP deficiency and AQP polarization to the leading edge of migrating cells, a mechanism of AQP-facilitated cell migration was proposed by Verkman and collaborators.⁹ According to this model, actin cleavage and ion uptake at the tip of lamellipodium creates local osmotic gradients and drives water influx, facilitating lamellipodial extension and cell migration.⁹ AQP-facilitated cell migration has also been found in brain astroglial cells,^10,11 kidney proximal tube cells¹² and skin cells.¹³ In this connection, AQP1 has been proposed as a novel promoter of tumor angiogenesis.¹⁴ It is still unclear, however, how actin is cleaved. On the other hand, according to Verkman’s model, AQP1 is the water channel that drives water influx.We have recently proposed a new model. In a recent paper published in PLoS ONE Journal, we have investigated the possi-ble relationship between AQP1 and the cytoskeleton in endothelial and melanoma cells (both expressing AQP1), focusing on the possible involvement of Lin proteins.¹⁵ The latter are plasma membrane-associated proteins containing one or several PDZ domains¹⁶ and are required for the organization of the cytoskeleton. A scaffold complex common for epithelial and neuronal cells is the heterotrimeric complex consisting of the CASK/Lin-2, Lin-7 and Lin-10 PDZ proteins.^17–20 In mammals, Lin-7 can recruit cell adhesion molecules, receptors, ion channels and signaling proteins.^17–20 Therefore, heterotrimeric PDZ complex plays a role in regulating the localization of interacting proteins. The novelties of our paper are the following: firstly, AQP1 plays the same role in human melanoma and endothelial cells, suggesting that this water channel has a global physiological role. Secondly, AQP1 interacts, at least, with Lin-7/β-catenin. Another interesting aspect is that the knock down of AQP1 induced the proteolytic degradation of Lin7/β-catenin through proteasoma complex. In the model proposed in PLoS ONE Journal, AQP1 is not only a water channel but a critical scaffold for plasma-membrane associated multiprotein-complex important for cytoskeleton build-up, adhesion and motility.¹⁵ Our data show, actually, that AQP1 plays a role in stabilizing the cytoskeleton affecting the migration capacity.²¹ Considering both Verkman’s model and our findings, I suggest that, in presence of local osmotic gradients like as at the tip of lamelllipodium, water is driven inside through AQP(s), leading to the disruption of scaffold proteins which are degraded through proteasoma (Lin7/β-catenin). The effect on the cell is the cleavage of actin.These findings corroborate the analysis of manifold cellular functions of AQPs in normal cells and in diseases and the possi-bility to consider aquaporins as specific therapeutic targets for various pathophysiological conditions.²² In particular, AQP1 might be an interesting target for tumors. In fact, AQP1 is expressed both by tumor and endothelial cells and a targeted inhibition or silencing of such a protein might affect both the migratory and the angiogenesis/vasculogenic mimicry capacity.Vasculogenic mimicry was described for the first time by the unique ability of aggressive melanoma cells to express an endothelial phenotype and to form vessel-like networks in three dimensional cultures, “mimicking” the pattern of embryonic vascular networks and recapitulating the patterned networks seen in patients with aggressive tumors correlated with poor prognosis (reviewed in ref. ²³). In fact, the word “vasculogenic” was selected to indicate the generation of the pathway de novo and “mimicry” was used because the tumor uses cell pathways for transporting fluid in tissues that were clearly not blood vessels. Additional studies have reported vasculogenic mimicry in several other tumor types (reviewed in ref. ²³). As shown in Figure 1, human melanoma cell line WM115 expresses AQP1 at the plasma membrane in vitro and only a few cells express such a water channel in tumor xenograft according to the low/undetectable number of initiating/cancer stem cells found in tumor xenograft and melanoma biopsies.²⁴Open in a separate windowFigure 1(A) Immunofluorescence of AQP1 in WM115 cells. Subconfluent cells were grown on 400 mm² glass cover and fixed in 4% paraformaldeide for 20 min. Thus, the cells were incubated with the same buffer containing 0.5% TRITON X-100 for 5 min and incubated in 1% BSA-PBS for 20 min and with the primary antibody (anti-AQP-1) overnight at 4°C. A secondary antibody conjugated with TRIC was used. Nuclei were stained with DAPI. (B) 5 × 10⁴ living WM115 cells were injected subcutaneous in SCID mice and the tumor mass was collected 19 days after and processed for immunohistochemistry. The slides (4–5 mm) deparaffinized were incubated overnight with anti-AQP1 (1:100) and the colour was developed using VIP. Magnification 200X. E, endothelial cells AQP1-positive. (C) Shows a higher magnification of a part of figure shown in (B) where few melanoma cells are AQP1-positive (1,000X).Finally, there are no AQP inhibitors reported that are suitable candidates for clinical development. An interesting new way might be to study the possibility of functionally significant AQP polymorphisms. In this connection, AQP4 polymorphisms was found to be associated with increased severity of brain edema.²⁵ It may be worthwhile to investigate polymorphisms of AQP1 or other AQPs in cancer and endothelial associated tumor.     

Impaired plasma nitric oxide availability and extracellular superoxide dismutase activity in healthy humans with advancing age

This study is aimed to verify the modifications of extracellular superoxide dismutase (EC-SOD) activity and its potential involvement on the mechanism responsible for the impairment of plasma nitric oxide (NO) availability occurring with advancing age in healthy humans. For this purpose, plasma samples were drawn from 40 healthy men, aged 20-92 years, in fasting state and used for measurements of stable end-product nitrite/nitrate (NOx), as expression of NO availability, EC-SOD activity, thiobarbituric acid reactive substances (TBARS) as marker of lipid peroxidation, Trolox equivalent antioxidant capacity (TEAC) as a measure of plasma total antioxidant capacity, and in vitro susceptibility of low density lipoprotein (LDL) to copper-mediated oxidation, evaluated as lag time. As indicated by our results, advancing age was significantly related to decreased plasma values of NOx (r = -0.877, P < 0.001), EC-SOD activity (r = -0.888, P < 0.001), TEAC (r = -0.647, P < 0.001) and lag time (r = -0.621, P < 0.001) as well as to an increased plasma amount of TBARS (r = 0.858, P < 0.001). NOx plasma level resulted independently predicted by EC-SOD activity and age. EC-SOD activity, in turn, was determined by age and TEAC. Taken together, findings of the present study give further insight into the mechanism related to age-associated endothelial dysfunction, indicating that the decreased EC-SOD activity may be involved in the progressive reduction of plasma NO availability with advancing age through the age-related impairment of oxidant/antioxidant balance.     

Effects of XeCl UV308 nm laser radiation on survival and mutability of recA-proficient and recA-defective Escherichia coli strains

recA1, recA13 and recA56 are considered null alleles of the Escherichia coli recA gene because they were shown to have essentially no activity in vivo. In this study, we used strains harboring the recA null alleles and their recA-proficient congenic counterpart to assess the lethal and the mutagenic effects elicited by near-UV(308 nm) coherent radiation generated by a XeCl excimer laser. We compared these effects with those produced by a conventional far-UV(254 nm) germicidal lamp. Compared to the germicidal lamp, the excimer laser was able to better discriminate the different recA-defective strains on the basis of their UV-radiation sensitivity, which was progressively higher in the strains with the alleles in the order recA1, recA56 and recA13. This finding was consistent with previous data on residual biochemical activities of the respective mutated RecA proteins in vitro. The discrepancy between the results obtained with the lamp and laser irradiation suggested that the biological response to the two radiations involves distinct mechanisms. This hypothesis was supported by the evidence that exposure to near-UV(308 nm) radiation induced mutagenesis in recA-defective strains at an extent considerably greater than in recA-proficient strains. In contrast, far-UV(254 nm)-radiation-induced mutagenesis was reported to be largely dependent on a functional recA allele.