Recombinant Human TSH Fails to Induce the Proliferation and Migration of Papillary Thyroid Carcinoma Cell Lines.

Thyrotropin (TSH) suppression is required in the management of patients with papillary thyroid carcinoma (PTC) to improve their outcomes, inevitably causing iatrogenic thyrotoxicosis. Nevertheless, the evidence supporting this practice remains limited and weak, and in vitro studies examining the mitogenic effects of TSH in cancerous cells used supraphysiological doses of bovine TSH, which produced conflicting results. Our study explores, for the first time, the impact of human recombinant thyrotropin (rh-TSH) on human PTC cell lines (K1 and TPC-1) that were transformed to overexpress the thyrotropin receptor (TSHR). The cells were treated with escalating doses of rh-TSH under various conditions, such as the presence or absence of insulin. The expression levels of TSHR and thyroglobulin (Tg) were determined, and subsequently, the proliferation and migration of both transformed and non-transformed cells were assessed. Under the conditions employed, rh-TSH was not adequate to induce either the proliferation or the migration rate of the cells, while Tg expression was increased. Our experiments indicate that clinically relevant concentrations of rh-TSH cannot induce proliferation and migration in PTC cell lines, even after the overexpression of TSHR. Further research is warranted to dissect the underlying molecular mechanisms, and these results could translate into better management of treatment for PTC patients.

Poly-Unsaturated Fatty Acids (PUFAs) from Cunninghamella elegans Grown on Glycerol Induce Cell Death and Increase Intracellular Reactive Oxygen Species.

Cunninghamella elegans NRRL-1393 is an oleaginous fungus able to synthesize and accumulate unsaturated fatty acids, amongst which the bioactive gamma-linolenic acid (GLA) has potential anti-cancer activities. C. elegans was cultured in shake-flask nitrogen-limited media with either glycerol or glucose (both at ≈60 g/L) employed as the sole substrate. The assimilation rate of both substrates was similar, as the total biomass production reached 13.0-13.5 g/L, c. 350 h after inoculation (for both instances, c. 27-29 g/L of substrate were consumed). Lipid production was slightly higher on glycerol-based media, compared to the growth on glucose (≈8.4 g/L vs. ≈7.0 g/L). Lipids from C. elegans grown on glycerol, containing c. 9.5% w/w of GLA, were transformed into fatty acid lithium salts (FALS), and their effects were assessed on both human normal and cancerous cell lines. The FALS exhibited cytotoxic effects within a 48 h interval with an IC50 of about 60 µg/mL. Additionally, a suppression of migration was shown, as a significant elevation of oxidative stress levels, and the induction of cell death. Elementary differences between normal and cancer cells were not shown, indicating a generic mode of action; however, oxidative stress level augmentation may increase susceptibility to anticancer drugs, improving chemotherapy effectiveness.

Autophagy and oxidative stress modulation mediate Bortezomib resistance in prostate cancer.

Proteasome inhibitors such as Bortezomib represent an established type of targeted treatment for several types of hematological malignancies, including multiple myeloma, Waldenstrom's macroglobulinemia, and mantle cell lymphoma, based on the cancer cell's susceptibility to impairment of the proteasome-ubiquitin system. However, a major problem limiting their efficacy is the emergence of resistance. Their application to solid tumors is currently being studied, while simultaneously, a wide spectrum of hematological cancers, such as Myelodysplastic Syndromes show minimal or no response to Bortezomib treatment. In this study, we utilize the prostate cancer cell line DU-145 to establish a model of Bortezomib resistance, studying the underlying mechanisms. Evaluating the resulting resistant cell line, we observed restoration of proteasome chymotrypsin-like activity, regardless of drug presence, an induction of pro-survival pathways, and the substitution of the Ubiquitin-Proteasome System role in proteostasis by induction of autophagy. Finally, an estimation of the oxidative condition of the cells indicated that the resistant clones reduce the generation of reactive oxygen species induced by Bortezomib to levels even lower than those induced in non-resistant cells. Our findings highlight the role of autophagy and oxidative stress regulation in Bortezomib resistance and elucidate key proteins of signaling pathways as potential pharmaceutical targets, which could increase the efficiency of proteasome-targeting therapies, thus expanding the group of molecular targets for neoplastic disorders.

Lower-Risk Myelodysplastic Syndrome (MDS) Patients Exhibit Diminished Proteasome Proteolytic Activity and High Intracellular Reactive Oxygen Species (ROS) Levels.

Myelodysplastic syndromes (MDS) constitute a heterogeneous group of clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis and an elevated risk of transformation to acute myeloid leukemia (AML). Available disease-modifying treatment approaches are limited. The ineffectiveness of proteasome inhibitors (PIs) in MDS patients is currently investigated, although it is unclear whether they rapidly develop resistance to PIs or whether proteasome proteolytic activity (PPA) is constitutively lower in the hematopoietic cells of these patients, thus limiting treatment effectiveness. We investigated 20 patients with MDS, categorized according to the International Prognostic Scoring System (IPSS) into a lower- or a higher-risk group. Peripheral blood mononuclear cells, bone marrow mononuclear cells, and cluster of differentiation 34-positive (CD34+) cells were isolated and assessed for the chymotrypsin-like activity of the proteasome and ß5 subunit accumulation. Additionally, intracellular reactive oxygen species (ROS) generation was screened. The lower-risk patient group (n=10) exhibited significantly lower proteasome activity (p<0.001) compared to both the higher-risk group (n=10) and healthy subjects (n=10). Furthermore, the lower-risk group had elevated oxidative stress levels (p<0.0001) and reduced ß5 subunit expression (p=0.0286). Both parameters were shown to be associated with transfusion dependency, since transfusion-dependent patients (n=5 in each subgroup) had decreased proteasome activity and simultaneously exhibited higher ROS levels. Our results indicate that reduced ß5 expression might potentially explain PIs' ineffectiveness in lower-risk MDS, elucidating the importance of the risk group in the selection of the proper treatment algorithm.

Albumin upregulates eNOS mRNA through ETRA/B in human proximal tubular epithelial cells.

BACKGROUND: Proximal tubular cells respond to proteinuria by expressing several cytokines and inflammatory molecules that induce interstitial fibrosis. Increased attention has been drawn toward the systems of endothelin (ET) and nitric oxide (NO). This work contributes to the elucidation of the interplay between these two systems in proximal tubular epithelial cells (PTECs) after exposure in proteinuric conditions. METHODS: HK-2 cells, a human PTEC line, were incubated with albumin, simulating proteinuric conditions. Cells were then lysed and either total RNA was isolated or whole cell extracts were prepared. PreproET-1, ET receptors (ETRA and ETRB) and NO synthases (eNOS, iNOS) mRNA accumulation was estimated by RT-PCR, and proteins by Western blot analysis. NO production was assessed using Griess reaction. Furthermore, we treated HK-2 cells with NO donor sodium nitroprusside, NO inhibitor L-NAME, ETRA inhibitor BQ123, ETRB inhibitor BQ788 and purified ET-1, and investigated the potential interplay between albumin-induced stimulation of NO or ET-1 systems. RESULTS: We found that albumin upregulates preproET-1, ETRA, ETRB, eNOS and iNOS mRNA as well as protein and stimulates NO production. Additionally, we recorded an ETRA/B dependent regulation of albumin-induced eNOS expression. CONCLUSIONS: For the first time an in vitro albumin-induced ET-1 and NO interplay was revealed.

The pseudopeptide HB-19 binds to cell surface nucleolin and inhibits angiogenesis.

BACKGROUND: Nucleolin is a protein over-expressed on the surface of tumor and endothelial cells. Recent studies have underlined the involvement of cell surface nucleolin in tumor growth and angiogenesis. This cell surface molecule serves as a receptor for various ligands implicated in pathophysiological processes such as growth factors, cell adhesion molecules like integrins, selectins or laminin-1, lipoproteins and viruses (HIV and coxsackie B). HB-19 is a synthetic multimeric pseudopeptide that binds cell surface expressed nucleolin and inhibits both tumor growth and angiogenesis. METHODOLOGY/PRINCIPAL FINDINGS: In the present work, we further investigated the biological actions of pseudopeptide HB-19 on HUVECs. In a previous work, we have shown that HB-19 inhibits the in vivo angiogenesis on the chicken embryo CAM assay. We now provide evidence that HB-19 inhibits the in vitro adhesion, migration and proliferation of HUVECs without inducing their apoptosis. The above biological actions seem to be regulated by SRC, ERK1/2, AKT and FAK kinases as we found that HB-19 inhibits their activation in HUVECs. Matrix metalloproteinases (MMPs) play crucial roles in tumor growth and angiogenesis, so we investigated the effect of HB-19 on the expression of MMP-2 and we found that HB-19 downregulates MMP-2 in HUVECs. Finally, down regulation of nucleolin using siRNA confirmed the implication of nucleolin in the biological actions of these peptides. CONCLUSIONS/SIGNIFICANCE: Taken together, these results indicate that HB-19 could constitute an interesting tool for tumor therapy strategy, targeting cell surface nucleolin.

Structure-activity studies of lGnRH-III through rational amino acid substitution and NMR conformational studies.

Lamprey gonadotropin-releasing hormone type III (lGnRH-III) is an isoform of GnRH isolated from the sea lamprey (Petromyzon marinus) with negligible endocrine activity in mammalian systems. Data concerning the superior direct anticancer activity of lGnRH-III have been published, raising questions on the structure-activity relationship. We synthesized 21 lGnRH-III analogs with rational amino acid substitutions and studied their effect on PC3 and LNCaP prostate cancer cell proliferation. Our results question the importance of the acidic charge of Asp6 for the antiproliferative activity and indicate the significance of the stereochemistry of Trp in positions 3 and 7. Furthermore, conjugation of an acetyl-group to the side chain of Lys8 or side chain cyclization of amino acids 1-8 increased the antiproliferative activity of lGnRH-III demonstrating that the proposed salt bridge between Asp6 and Lys8 is not crucial. Conformational studies of lGnRH-III were performed through NMR spectroscopy, and the solution structure of GnRH-I was solved. In solution, lGnRH-III adopts an extended backbone conformation in contrast to the well-defined ß-turn conformation of GnRH-I.

Nucleolin mediates the antiangiogenesis effect of the pseudopeptide N6L.

BACKGROUND: Nucleolin is a protein over-expressed on the surface of activated cells. Recent studies have underlined the involvement of cell surface nucleolin in angiogenesis processes. This cell surface molecule serves as a receptor for various ligands implicated in pathophysiological processes such as growth factors, cell adhesion molecules like integrins, selectins or laminin-1, lipoproteins and viruses. N6L is a synthetic multimeric pseudopeptide that binds cell surface expressed nucleolin and inhibits cell proliferation. RESULTS: In the present work, we further investigated the mechanisms of action of pseudopeptide N6L on angiogenesis using HUVECs. We provide evidence that N6L inhibits the in vitro adhesion, proliferation and migration of HUVECs without inducing their apoptosis. In addition, we found that N6L downregulates MMP-2 in HUVECs. The above biological actions are regulated by SRC, ERK1/2, AKT and FAK kinases as we found that N6L inhibits their activation in HUVECs. Finally, down regulation of nucleolin using siRNA demonstrated the implication of nucleolin in the biological actions of these peptides. CONCLUSIONS: Taken together, these results indicate that N6L could constitute an interesting therapeutic tool for treating diseases associated with excessive angiogenesis.

Loss of receptor protein tyrosine phosphatase ß/ζ (RPTPß/ζ) promotes prostate cancer metastasis.

BACKGROUND: The role of pleiotrophin and its receptors RPTPß/ζ and Syndecan-3 during tumor metastasis remains unknown. RESULTS: RPTPß/ζ knockdown initiates EMT, promotes pleiotrophin-mediated migration and attachment through Syndecan-3 and induces in vivo metastasis. CONCLUSION: RPTPß/ζ plays a suppressor-like role in prostate cancer metastasis. SIGNIFICANCE: Boosting RPTPß/ζ or attenuating Syndecan-3 signaling pathways may lead to more effective therapeutic strategies in treating prostate cancer metastasis. Pleiotrophin is a growth factor that induces carcinogenesis. Despite the fact that many published reports focused on the role of pleiotrophin and its receptors, receptor protein tyrosine phosphatase (RPTPß/ζ), and syndecan-3 during tumor development, no information is available regarding their function in tumor metastasis. To investigate the mechanism through which pleiotrophin regulates tumor metastasis, we used two different prostate carcinoma cell lines, DU145 and PC3, in which the expression of RPTPß/ζ or syndecan-3 was down-regulated by the RNAi technology. The loss of RPTPß/ζ expression initiated epithelial-to-mesenchymal transition (EMT) and increased the ability of the cells to migrate and invade. Importantly, the loss of RPTPß/ζ expression increased metastasis in nude mice in an experimental metastasis assay. We also demonstrate that RPTPß/ζ counterbalanced the pleiotrophin-mediated syndecan-3 pathway. While the inhibition of syndecan-3 expression inhibited the pleiotrophin-mediated cell migration and attachment through the Src and Fak pathway, the inhibition of RPTPß/ζ expression increased pleiotrophin-mediated migration and attachment through an interaction with Src and the subsequent activation of a signal transduction pathway involving Fak, Pten, and Erk1/2. Taken together, these results suggest that the loss of RPTPß/ζ may contribute to the metastasis of prostate cancer cells by inducing EMT and promoting pleiotrophin activity through the syndecan-3 pathway.

Hemodialysis removes uremic toxins that alter the biological actions of endothelial cells.

Chronic kidney disease is linked to systemic inflammation and to an increased risk of ischemic heart disease and atherosclerosis. Endothelial dysfunction associates with hypertension and vascular disease in the presence of chronic kidney disease but the mechanisms that regulate the activation of the endothelium at the early stages of the disease, before systemic inflammation is established remain obscure. In the present study we investigated the effect of serum derived from patients with chronic kidney disease either before or after hemodialysis on the activation of human endothelial cells in vitro, as an attempt to define the overall effect of uremic toxins at the early stages of endothelial dysfunction. Our results argue that uremic toxins alter the biological actions of endothelial cells and the remodelling of the extracellular matrix before signs of systemic inflammatory responses are observed. This study further elucidates the early events of endothelial dysfunction during toxic uremia conditions allowing more complete understanding of the molecular events as well as their sequence during progressive renal failure.

Pleiotrophin expression and role in physiological angiogenesis in vivo: potential involvement of nucleolin.

BACKGROUND: Pleiotrophin (PTN) is a heparin-binding growth factor with significant role(s) in tumour growth and angiogenesis. Although implication of endogenous PTN has been studied in several in vivo models of tumour angiogenesis, its role in physiological angiogenesis has not been addressed. In the present work, we studied expression and functional significance of endogenous PTN during angiogenesis in the chicken embryo chorioallantoic membrane (CAM). METHODS: Using molecular, cellular and biochemical assays, we studied the expression pattern of PTN in CAM and human endothelial cells and its possible interaction with nucleolin (NCL). CAM cells were transfected with a pCDNA3.1 vector, empty (PC) or containing full length cDNA for PTN in antisense orientation (AS-PTN). Angiogenesis was estimated by measuring total vessel length. In vitro, human endothelial cells migration was studied by using a transwell assay, and down-regulation of NCL was performed by using a proper siRNA. RESULTS: Endogenous PTN mRNA and protein levels, as well as protein levels of its receptor protein tyrosine phosphatase beta/zeta (RPTPß/ζ) were maximal at early stages, when CAM angiogenesis is active. Application of AS-PTN onto CAM at days of active angiogenesis was not toxic to the tissue and led to dose-dependent decreased expression of endogenous PTN, ERK1/2 activity and angiogenesis. Interestingly, endogenous PTN was also immunolocalized at the endothelial cell nucleus, possibly through interaction with NCL, a protein that has a significant role in the nuclear translocation of many proteins. Down-regulation of NCL by siRNA in human endothelial cells significantly decreased nuclear PTN, verifying this hypothesis. Moreover, it led to abolishment of PTN-induced endothelial cell migration, suggesting, for the first time, that PTN-NCL interaction has a functional significance. CONCLUSIONS: Expression of endogenous PTN correlates with and seems to be involved in angiogenesis of the chicken embryo CAM. Our data suggest that NCL may have a role, increasing the number of growth factors whose angiogenic/tumorigenic activities are mediated by NCL.

A simple approach to cancer therapy afforded by multivalent pseudopeptides that target cell-surface nucleoproteins.

Recent studies have implicated the involvement of cell surface forms of nucleolin in tumor growth. In this study, we investigated whether a synthetic ligand of cell-surface nucleolin known as N6L could exert antitumor activity. We found that N6L inhibits the anchorage-dependent and independent growth of tumor cell lines and that it also hampers angiogenesis. Additionally, we found that N6L is a proapoptotic molecule that increases Annexin V staining and caspase-3/7 activity in vitro and DNA fragmentation in vivo. Through affinity isolation experiments and mass-spectrometry analysis, we also identified nucleophosmin as a new N6L target. Notably, in mouse xenograft models, N6L administration inhibited human tumor growth. Biodistribution studies carried out in tumor-bearing mice indicated that following administration N6L rapidly localizes to tumor tissue, consistent with its observed antitumor effects. Our findings define N6L as a novel anticancer drug candidate warranting further investigation.

A peptide corresponding to the C-terminal region of pleiotrophin inhibits angiogenesis in vivo and in vitro.

Pleiotrophin (PTN) is a heparin-binding growth factor that plays a significant role in tumor growth and angiogenesis. We have previously shown that in order for PTN to induce migration of endothelial cells, binding to both α(ν) ß(3) integrin and its receptor protein tyrosine phosphatase beta/zeta (RPTPß/ζ) is required. In the present study we show that a synthetic peptide corresponding to the last 25 amino acids of the C-terminal region of PTN (PTN(112-136) ) inhibited angiogenesis in the in vivo chicken embryo chorioallantoic membrane (CAM) assay and PTN-induced migration and tube formation of human endothelial cells in vitro. PTN(112-136) inhibited binding of PTN to α(ν) ß(3) integrin, and as shown by surface plasmon resonance (SPR) measurements, specifically interacted with the specificity loop of the extracellular domain of ß(3) . Moreover, it abolished PTN-induced FAK Y397 phosphorylation, similarly to the effect of a neutralizing α(ν) ß(3) -selective antibody. PTN(112-136) did not affect binding of PTN to RPTPß/ζ in endothelial cells and induced ß(3) Y773 phosphorylation and ERK1/2 activation to a similar extent with PTN. This effect was inhibited by down-regulation of RPTPß/ζ by siRNA or by c-src inhibition, suggesting that PTN(112-136) may interact with RPTPß/ζ. NMR spectroscopy studies showed that PTN(112-136) was characterized by conformational flexibility and absence of any element of secondary structure at room temperature, although the biologically active peptide segment 123-132 may adopt a defined structure at lower temperature. Collectively, our data suggest that although PTN(112-136) induces some of the signaling pathways triggered by PTN, it inhibits PTN-induced angiogenic activities through inhibition of PTN binding to α(ν) ß(3) integrin.

Enzymatic stability, solution structure, and antiproliferative effect on prostate cancer cells of leuprolide and new gonadotropin-releasing hormone peptide analogs.

Analogs of GnRH, including [DLeu6, desGly1o]-GnRH-NHEt (leuprolide, commercial product), have been widely used in oncology to induce reversible chemical castration. Several studies have provided evidence that, besides their pituitary effects, GnRH analogs may exert direct antiproliferative effects on tumor cells. To study the effect of modifications in positions 4 and 6 of leuprolide on prostate cancer cell proliferation, we synthesized 12 new leuprolide analogs. All GnRH analogs lacked the carboxy-terminal Gly10-amide of GnRH, and an ethylamide residue was added to Pro9. Gly6 was substituted by DLys, Nepsilon-modified DLys, Glu, and DGlu. To improve the enzymatic stability, NMeSer was incorporated in position 4, and the rate of hydrolysis by alpha-chymotrypsin and subtilisin was investigated. Our results demonstrate that this incorporation increases enzymatic stability in all analogs of GnRH, whereas the antiproliferative effect on PC3 and LNCaP prostate cancer cells is similar to that of leuprolide. Conformational studies were performed to elucidate structural changes occurring on substitution of native residues and to study structure-activity relationship for these analogs. The solution models of [DLeu6, desGly10]-GnRH-NHEt (leuprolide), [NMeSer4, DGlu6, desGly10]-GnRH-NHEt, [Glu6, desGly10]-GnRH-NHEt, and [DGIu6, desGly10]-GnRH-NHEt peptides were determined through two-dimensional nuclear magnetic resonance spectroscopy in dimethylsulfoxide. Nuclear magnetic resonance data provide experimental evidence for the U-turn-like structure appeared in all four analogs, which could be characterized as beta-hairpin conformation. The most stable analog [NMeSer4, DGlu6, desGly10]-GnRH-NHEt against proteolytic cleavage forms a second extra backbone turn observed for residues 1-4.

A Pleiotrophin C-terminus peptide induces anti-cancer effects through RPTPß/ζ.

BACKGROUND: Pleiotrophin, also known as HARP (Heparin Affin Regulatory Peptide) is a growth factor expressed in various tissues and cell lines. Pleiotrophin participates in multiple biological actions including the induction of cellular proliferation, migration and angiogenesis, and is involved in carcinogenesis. Recently, we identified and characterized several pleiotrophin proteolytic fragments with biological activities similar or opposite to that of pleiotrophin. Here, we investigated the biological actions of P(122-131), a synthetic peptide corresponding to the carboxy terminal region of this growth factor. RESULTS: Our results show that P(122-131) inhibits in vitro adhesion, anchorage-independent proliferation, and migration of DU145 and LNCaP cells, which express pleiotrophin and its receptor RPTPß/ζ. In addition, P(122-131) inhibits angiogenesis in vivo, as determined by the chicken embryo CAM assay. Investigation of the transduction mechanisms revealed that P(122-131) reduces the phosphorylation levels of Src, Pten, Fak, and Erk1/2. Finally, P(122-131) not only interacts with RPTPß/ζ, but also interferes with other pleiotrophin receptors, as demonstrated by selective knockdown of pleiotrophin or RPTPß/ζ expression with the RNAi technology. CONCLUSIONS: In conclusion, our results demonstrate that P(122-131) inhibits biological activities that are related to the induction of a transformed phenotype in PCa cells, by interacing with RPTPß/ζ and interfering with other pleiotrophin receptors. Cumulatively, these results indicate that P(122-131) may be a potential anticancer agent, and they warrant further study of this peptide.

Interaction of endothelin-1 and nitric oxide pathways in human tubular epithelial cells under the influence of cyclosporine-A.

BACKGROUND: The exact mechanism of cyclosporine (CsA) nephrotoxicity has not been clarified. In this study, we investigated the effect of pharmacological doses of CsA on the production of nitric oxide synthases (NOSs) and endothelin (ET) receptors (ETR-A, ETR-B), in human tubular cells [human kidney (HK)-2], to identify any implication of these pathways in CsA nephrotoxicity. METHODS: Human tubular epithelial cells (HK-2) were cultured in the presence of CsA at various concentrations (0-1000 ng/mL). Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to determine mRNA synthesis of NOSs (eNOS, iNOS) and ET receptors (ETR-A, ETR-B) and western blot analysis for the subsequent proteins. RESULTS: A dose-dependent induction of synthesis of NO synthases eNOS and iNOS and ET receptors ETR-A and ETR-B was observed, even at therapeutic doses of CsA. An interaction between NO and ET-1 systems under the influence of CsA was also observed. Blockage of NO production was followed by down-regulation of ETR-B whereas blockade of ET pathway with ET receptor antagonists was followed by down-regulation of eNOS expression. CONCLUSION: CsA induces NOSs as well as ET receptor mRNA and protein synthesis in tubular epithelial cells. The up-regulation of NO and ET-1 pathways is probably implicated in the nephrotoxic action of CsA, whereas an interplay between ETR-B and eNOS seems to be involved.

Cyclosporine induces endothelin-1 mRNA synthesis and nitric oxide production in human proximal tubular epithelial cell cultures.

BACKGROUND: Cyclosporine (CsA) is implicated in the development of chronic allograft nephropathy, which is related to reduced long-term allograft survival. The activation of tubular epithelial cells is involved in the renal scarring process via stimulation of factors such as endothelin-1 (ET-1) and nitric oxide (NO). The effect of CsA on the activation of tubular epithelial cells towards increased production of ET-1 and NO was investigated in this study. METHODS: Human tubular epithelial cells (HK-2) were cultured in the presence of CsA at different concentrations (125, 250, 500, and 1,000 ng/mL). ET-1 m-RNA and NO production were measured using RT-PCR and Griess method, respectively. The cytotoxic effect of CsA was examined by the MTT method and cell count. RESULTS: A statistically significant and dose-dependent cytotoxic effect of cyclosporine on HK-2 cells was observed. A dose-dependent up-regulation of ET-1 mRNA production and NO accumulation was observed under the influence of CsA. CONCLUSION: Increased synthesis of endothelin-1 mRNA and nitric oxide as well as a significant cytotoxic effect on tubular epithelial cells under the influence of CsA might be related to the development of CsA nephrotoxicity.

Suppression of tumor growth and angiogenesis by a specific antagonist of the cell-surface expressed nucleolin.

BACKGROUND: Emerging evidences suggest that nucleolin expressed on the cell surface is implicated in growth of tumor cells and angiogenesis. Nucleolin is one of the major proteins of the nucleolus, but it is also expressed on the cell surface where is serves as a binding protein for variety of ligands implicated in cell proliferation, differentiation, adhesion, mitogenesis and angiogenesis. METHODOLOGY/PRINCIPAL FINDINGS: By using a specific antagonist that binds the C-terminal tail of nucleolin, the HB-19 pseudopeptide, here we show that the growth of tumor cells and angiogenesis are suppressed in various in vitro and in vivo experimental models. HB-19 inhibited colony formation in soft agar of tumor cell lines, impaired migration of endothelial cells and formation of capillary-like structures in collagen gel, and reduced blood vessel branching in the chick embryo chorioallantoic membrane. In athymic nude mice, HB-19 treatment markedly suppressed the progression of established human breast tumor cell xenografts in nude mice, and in some cases eliminated measurable tumors while displaying no toxicity to normal tissue. This potent antitumoral effect is attributed to the direct inhibitory action of HB-19 on both tumor and endothelial cells by blocking and down regulating surface nucleolin, but without any apparent effect on nucleolar nucleolin. CONCLUSION/SIGNIFICANCE: Our results illustrate the dual inhibitory action of HB-19 on the tumor development and the neovascularization process, thus validating the cell-surface expressed nucleolin as a strategic target for an effective cancer drug. Consequently, the HB-19 pseudopeptide provides a unique candidate to consider for innovative cancer therapy.

Heparin affin regulatory peptide/pleiotrophin mediates fibroblast growth factor 2 stimulatory effects on human prostate cancer cells.

Fibroblast growth factor 2 (FGF2) is a pleiotropic growth factor that has been implicated in prostate cancer formation and progression. In the present study we found that exogenous FGF2 significantly increased human prostate cancer LNCaP cell proliferation and migration. Heparin affin regulatory peptide (HARP) or pleiotrophin seems to be an important mediator of FGF2 stimulatory effects, since the latter had no effect on stably transfected LNCaP cells that did not express HARP. Moreover, FGF2, through FGF receptors (FGFRs), significantly induced HARP expression and secretion by LNCaP cells and increased luciferase activity of a reporter gene vector carrying the full-length promoter of HARP gene. Using a combination of Western blot analyses, as well as genetic and pharmacological inhibitors, we found that activation of FGFR by FGF2 in LNCaP cells leads to NAD(P)H oxidase-dependent hydrogen peroxide production, phosphorylation of ERK1/2 and p38, activation of AP-1, increased expression and secretion of HARP, and, finally, increased cell proliferation and migration. These results establish the role and the mode of activity of FGF2 in LNCaP cells and support an interventional role of HARP in FGF2 effects, providing new insights on the interplay among growth factor pathways within prostate cancer cells.

Antioxidants modify the effect of X rays on blood vessels.

BACKGROUND: It was recently shown, with the chicken embryo chorioallantoic membrane (CAM) model, that X rays decrease the number of blood vessels within the first hours after irradiation. In the present study, the possible role of reactive oxygen species (ROS) and nitric oxide (NO) in this effect of X rays was evaluated. MATERIALS AND METHODS: An area of 1 cm2 of the CAM, restricted by a plastic ring, was irradiated at room temperature, in the presence or absence of the tested agents. The number of vessels was measured 48 h after irradiation of the tissue. RESULTS: Superoxide dismutase and tempol, which are superoxide ion scavengers and catalase, a hydrogen peroxide scavenger, had additive effects, while dimethylsulfoxide, a hydroxyl radical scavenger, reversed the vascular targeting effect of X rays. The combination of X rays with W1400, a selective inducible NO synthase (NOS) inhibitor, had an additive effect on the decrease in number of CAM blood vessels. In contrast, L-NAME, a non-selective NOS inhibitor and D-NAME, its inactive analog, reversed the vascular-targeting effect of X rays, possibly due to their ability to act as potent hydroxyl radical scavengers. CONCLUSION: The above data collectively suggest that hydroxyl radicals mediate the damaging effects of X rays on CAM blood vessels, while antioxidants against other ROS do not protect against the vascular-targeting effect of X rays.