Decoding NADPH oxidase 4 expression in human tumors.

NADPH oxidase 4 (NOX4) is a redox active, membrane-associated protein that contributes to genomic instability, redox signaling, and radiation sensitivity in human cancers based on its capacity to generate H2O2 constitutively. Most studies of NOX4 in malignancy have focused on the evaluation of a small number of tumor cell lines and not on human tumor specimens themselves; furthermore, these studies have often employed immunological tools that have not been well characterized. To determine the prevalence of NOX4 expression across a broad range of solid tumors, we developed a novel monoclonal antibody that recognizes a specific extracellular region of the human NOX4 protein, and that does not cross-react with any of the other six members of the NOX gene family. Evaluation of 20 sets of epithelial tumors revealed, for the first time, high levels of NOX4 expression in carcinomas of the head and neck (15/19 patients), esophagus (12/18 patients), bladder (10/19 patients), ovary (6/17 patients), and prostate (7/19 patients), as well as malignant melanoma (7/15 patients) when these tumors were compared to histologically-uninvolved specimens from the same organs. Detection of NOX4 protein upregulation by low levels of TGF-ß1 demonstrated the sensitivity of this new probe; and immunofluorescence experiments found that high levels of endogenous NOX4 expression in ovarian cancer cells were only demonstrable associated with perinuclear membranes. These studies suggest that NOX4 expression is upregulated, compared to normal tissues, in a well-defined, and specific group of human carcinomas, and that its expression is localized on intracellular membranes in a fashion that could modulate oxidative DNA damage.

Depletion of oxaloacetate decarboxylase FAHD1 inhibits mitochondrial electron transport and induces cellular senescence in human endothelial cells.

In this study we report the identification of FAH domain containing protein 1 (FAHD1), a recently described member of the fumarylacetoacetate hydrolase (FAH) superfamily of metabolic enzymes, as a novel player in the regulation of cellular senescence. FAHD1 was found in a proteomic screen searching for mitochondrial proteins, which are differentially regulated in mitochondria from young and senescent human endothelial cells, and subsequently identified as oxaloacetate decarboxylase. We report here that depletion of FAHD1 from human endothelial cells inhibited mitochondrial energy metabolism and subsequently induced premature senescence. Whereas senescence induced by FAHD1 depletion was not associated with DNA damage, we noted a reduction of mitochondrial ATP-coupled respiration associated with upregulation of the cdk inhibitor p21. These results indicate that FAHD1 is required for mitochondrial function in human cells and provide additional support to the growing evidence that mitochondrial dysfunction can induce cellular senescence by metabolic alterations independent of the DNA damage response pathway.

NADPH oxidase 4 deficiency increases tubular cell death during acute ischemic reperfusion injury.

NADPH oxidase 4 (NOX4) is highly expressed in kidney proximal tubular cells. NOX4 constitutively produces hydrogen peroxide, which may regulate important pro-survival pathways. Renal ischemia reperfusion injury (IRI) is a classical model mimicking human ischemic acute tubular necrosis. We hypothesized that NOX4 plays a protective role in kidney IRI. In wild type (WT) animals subjected to IRI, NOX4 protein expression increased after 24 hours. NOX4 KO (knock-out) and WT littermates mice were subjected to IRI. NOX4 KO mice displayed decreased renal function and more severe tubular apoptosis, decreased Bcl-2 expression and higher histologic damage scores compared to WT. Activation of NRF2 was decreased in NOX4 KO mice in response to IRI. This was related to decreased KEAP1 oxidation leading to decreased NRF2 stabilization. This resulted in decreased glutathione levels. In vitro silencing of NOX4 in cells showed an enhanced propensity to apoptosis, with reduced expression of NRF2, glutathione content and Bcl-2 expression, similar to cells derived from NOX4 KO mice. Overexpression of a constitutively active form of NRF2 (caNRF2) in NOX4 depleted cells rescued most of this phenotype in cultured cells, implying that NRF2 regulation by ROS issued from NOX4 may play an important role in its anti-apoptotic property.

A monoclonal antibody raised against bacterially expressed MPV17 sequences shows peroxisomal, endosomal and lysosomal localisation in U2OS cells.

Recessive mutations in the MPV17 gene cause mitochondrial DNA depletion syndrome, a fatal infantile genetic liver disease in humans. Loss of function in mice leads to glomerulosclerosis and sensineural deafness accompanied with mitochondrial DNA depletion. Mutations in the yeast homolog Sym1, and in the zebra fish homolog tra cause interesting, but not obviously related phenotypes, although the human gene can complement the yeast Sym1 mutation. The MPV17 protein is a hydrophobic membrane protein of 176 amino acids and unknown function. Initially localised in murine peroxisomes, it was later reported to be a mitochondrial inner membrane protein in humans and in yeast. To resolve this contradiction we tested two new mouse monoclonal antibodies directed against the human MPV17 protein in Western blots and immunohistochemistry on human U2OS cells. One of these monoclonal antibodies showed specific reactivity to a protein of 20 kD absent in MPV17 negative mouse cells. Immunofluorescence studies revealed colocalisation with peroxisomal, endosomal and lysosomal markers, but not with mitochondria. This data reveal a novel connection between a possible peroxisomal/endosomal/lysosomal function and mitochondrial DNA depletion.

ROS signaling by NADPH oxidase 5 modulates the proliferation and survival of prostate carcinoma cells.

Prostate cancer (PCa) is the most commonly diagnosed cancer and second leading cause of male cancer death in Western nations. Thus, new treatment modalities are urgently needed. Elevated production of reactive oxygen species (ROS) by NADPH oxidase (Nox) enzymes is implicated in tumorigenesis of the prostate and other tissues. However, the identity of the Nox enzyme(s) involved in prostate carcinogenesis remains largely unknown. Analysis of radical prostatectomy tissue samples and benign and malignant prostate epithelial cell lines identified Nox5 as an abundantly expressed Nox isoform. Consistently, immunohistochemical staining of a human PCa tissue microarray revealed distinct Nox5 expression in epithelial cells of benign and malignant prostatic glands. shRNA-mediated knockdown of Nox5 impaired proliferation of Nox5-expressing (PC-3, LNCaP) but not Nox5-negative (DU145) PCa cell lines. Similar effects were observed upon ROS ablation via the antioxidant N-acetylcysteine confirming ROS as the mediators. In addition, Nox5 silencing increased apoptosis of PC-3 cells. Concomitantly, protein kinase C zeta (PKCζ) protein levels and c-Jun N-terminal kinase (JNK) phosphorylation were reduced. Moreover, the effect of Nox5 knockdown on PC-3 cell proliferation could be mimicked by pharmacological inhibition of JNK. Collectively, these data indicate that Nox5 is expressed at functionally relevant levels in the human prostate and clinical PCa. Moreover, findings herein suggest that Nox5-derived ROS and subsequent depletion of PKCζ and JNK inactivation play a critical role in modulating intracellular signaling cascades involved in the proliferation and survival of PCa cells. © 2014 The Authors. Molecular Carcinogenesis published by Wiley Periodicals, Inc.

FAH domain containing protein 1 (FAHD-1) is required for mitochondrial function and locomotion activity in C. elegans.

The fumarylacetoacetate hydrolase (FAH) protein superfamily of metabolic enzymes comprises a diverse set of enzymatic functions, including ß-diketone hydrolases, decarboxylases, and isomerases. Of note, the FAH superfamily includes many prokaryotic members with very distinct functions that lack homologs in eukaryotes. A prokaryotic member of the FAH superfamily, referred to as Cg1458, was shown to encode a soluble oxaloacetate decarboxylase (ODx). Based on sequence homologies to Cg1458, we recently identified human FAH domain containing protein-1 (FAHD1) as the first eukaryotic oxaloacetate decarboxylase. The physiological functions of ODx in eukaryotes remain unclear. Here we have probed the function of fahd-1, the nematode homolog of FAHD1, in the context of an intact organism. We found that mutation of fahd-1 resulted in reduced brood size, a deregulation of the egg laying process and a severe locomotion deficit, characterized by a reduced frequency of body bends, reduced exploratory movements and reduced performance in an endurance exercise test. Notably, mitochondrial function was altered in the fahd-1(tm5005) mutant strain, as shown by a reduction of mitochondrial membrane potential and a reduced oxygen consumption of fahd-1(tm5005) animals. Mitochondrial dysfunction was accompanied by lifespan extension in worms grown at elevated temperature; however, unlike in mutant worms with a defect in the electron transport chain, the mitochondrial unfolded protein response was not upregulated in worms upon inactivation of fahd-1. Together these data establish a role of fahd-1 to maintain mitochondrial function and consequently physical activity in nematodes.

Alpha7 nicotinic receptor activation protects against oxidative stress via heme-oxygenase I induction.

Subchronic oxidative stress and inflammation are being increasingly implicated in the pathogenesis of numerous diseases, such as Alzheimer's or Parkinson's disease. This study was designed to evaluate the potential protective role of α7 nicotinic receptor activation in an in vitro model of neurodegeneration based on subchronic oxidative stress. Rat organotypic hippocampal cultures (OHCs) were exposed for 4 days to low concentration of lipopolysaccharide (LPS) and the complex III mitochondrial blocker, antimycin-A. Antimycin-A (0.1µM) and lipopolysaccharide (1ng/ml) caused low neurotoxicity on their own, measured as propidium iodide fluorescence in CA1 and CA3 regions. However, their combination (LPS/AA) caused a greater detrimental effect, in addition to mitochondrial depolarization, overproduction of reactive oxygen species (ROS) and Nox4 overexpression. Antimycin-A per se increased ROS and mitochondrial depolarization, although these effects were significantly higher when combined with LPS. More interesting was the finding that exposure of OHCs to the combination of LPS/AA triggered aberrant protein aggregation, measured as thioflavin S immunofluorescence. The α7 nicotinic receptor agonist, PNU282987, prevented the neurotoxicity and the pathological hallmarks observed in the LPS/AA subchronic toxicity model (oxidative stress and protein aggregates); these effects were blocked by α-bungarotoxin and tin protoporphyrin, indicating the participation of α7 nAChRs and heme-oxygenase I induction. In conclusion, subchronic exposure of OHCs to low concentration of antimycin-A plus LPS reproduced pathological features of neurodegenerative disorders. α7 nAChR activation ameliorated these alterations by a mechanism involving heme-oxygenase I induction.

Ultrastructural Morphometry Points to a New Role for LAMTOR2 in Regulating the Endo/Lysosomal System.

The late endosomal adaptor protein LAMTOR2/p14 is essential for tissue homeostasis by controlling MAPK and mTOR signaling, which in turn regulate cell growth and proliferation, migration and spreading. Moreover, LAMTOR2 critically controls architecture and function of the endocytic system, including epidermal growth factor receptor (EGFR) degradation in lysosomes, positioning of late endosomes and defense against intracellular pathogens. Here we describe the multifaceted ultrastructural phenotype of the endo/lysosomal system of LAMTOR2-deficient mouse embryonic fibroblasts. Quantitative (immuno-)electron microscopy of cryo-fixed samples revealed significantly reduced numbers of recycling tubules emanating from maturing multivesicular bodies (MVB). Instead, a distinct halo of vesicles surrounded MVB, tentatively interpreted as detached, jammed recycling tubules. These morphological changes in LAMTOR2-deficient cells correlated with the presence of growth factors (e.g. EGF), but were similarly induced in control cells by inactivating mTOR. Furthermore, proper transferrin receptor trafficking and recycling were apparently dependent on an intact LAMTOR complex. Finally, a severe imbalance in the relative proportions of endo/lysosomes was found in LAMTOR2-deficient cells, resulting from increased amounts of mature MVB and (autophago)lysosomes. These observations suggest that the LAMTOR/Ragulator complex is required not only for maintaining the homeostasis of endo/lysosomal subpopulations but also contributes to the proper formation of MVB-recycling tubules, and regulation of membrane/cargo recycling from MVB.

Identification of FAH domain-containing protein 1 (FAHD1) as oxaloacetate decarboxylase.

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins occur in both prokaryotes and eukaryotes, where they carry out diverse enzymatic reactions, probably related to structural differences in their respective FAH domains; however, the precise relationship between structure of the FAH domain and the associated enzyme function remains elusive. In mammals, three FAH domain-containing proteins, FAHD1, FAHD2A, and FAHD2B, are known; however, their enzymatic function, if any, remains to be demonstrated. In bacteria, oxaloacetate is subject to enzymatic decarboxylation; however, oxaloacetate decarboxylases (ODx) were so far not identified in eukaryotes. Based on molecular modeling and subsequent biochemical investigations, we identified FAHD1 as a eukaryotic ODx enzyme. The results presented here indicate that dedicated oxaloacetate decarboxylases exist in eukaryotes.

Analysis of human papillomavirus E7 protein status in C-33A cervical cancer cells.

High-risk human papillomaviruses (HPV) are the main etiologic factor for the development of cervical cancer. Infections by these viruses have been detected in virtually all cervical cancers. C-33A is one of the rare cervical cancer derived cell lines considered as HPV-negative. Employing monoclonal antibodies raised against a conformational epitope of the HPV-16 E7 oncoprotein, we present evidence suggesting that E7-positive cells can be sporadically and transiently detected in C-33A cell cultures. Immunoblotting with affinity-purified rabbit polyclonal anti-HPV 16 E7 antisera and q-RT-PCR analysis suggest that these cells do probably not express HPV-16 E7. Moreover, we show that the HPV E7 protein level differs considerably between individual cells in cultures of several established cervical cancer cell lines. Our data suggest that expression of the E7 protein is variable in established cervical cancer cell lines including C-33A cells.

Mitochondrial respiratory chain complex I is inactivated by NADPH oxidase Nox4.

ROS (reactive oxygen species) generated by NADPH oxidases play an important role in cellular signal transduction regulating cell proliferation, survival and differentiation. Nox4 (NADPH oxidase 4) induces cellular senescence in human endothelial cells; however, intracellular targets for Nox4 remained elusive. In the present study, we show that Nox4 induces mitochondrial dysfunction in human endothelial cells. Nox4 depletion induced alterations in mitochondrial morphology, stabilized mitochondrial membrane potential and decreased production of H(2)O(2) in mitochondria. High-resolution respirometry in permeabilized cells combined with native PAGE demonstrated that Nox4 specifically inhibits the activity of mitochondrial electron transport chain complex I, and this was associated with a decreased concentration of complex I subunits. These data suggest a new pathway by which sustained Nox4 activity decreases mitochondrial function.

Characterisation of Nox4 inhibitors from edible plants.

NADPH oxidases transport electrons from cytosolic NADPH through biological membranes to generate reactive oxygen species. NADPH oxidase 4, broadly expressed in humans, is an interesting pharmacological target, since its activity is deregulated in several diseases, including pulmonary fibrosis, diabetic nephropathy, and cardiac hypertrophy. Whereas several candidate NADPH oxidase 4 inhibitors were recently described, most of these compounds are either unspecific or toxic. Here we set out to identify new NADPH oxidase 4 inhibitors from edible plants, in an attempt to decrease the number of hits with toxic side effects. We screened a compound library prepared from edible plants for new bioactives with the ability to inhibit the activity of NADPH oxidase 4. Using both cell-based and cell-free assays, we identified several compounds with significant inhibitory activity towards NADPH oxidase 4. For selected compounds, the activity profile towards NADPH oxidase 2 and NADPH oxidase 5 was established, and controls were carried out to exclude general reactive oxygen species scavengers. A number of promising NADPH oxidase 4 inhibitors from edible plants was identified and characterised. Several new chemical entities are disclosed which act as NADPH oxidase 4 inhibitors, and the efficacies of our best hits, in particular several diarylheptanoids and lignans, are comparable to the best available pharmacological NADPH oxidase 4 inhibitors. These findings will provide valuable tools to study mechanisms of NADPH oxidase inhibition.

Analysis of the cellular uptake and nuclear delivery of insulin-like growth factor binding protein-3 in human osteosarcoma cells.

Insulin-like growth factor (IGF) binding protein-3 (IGFBP-3) regulates cell proliferation and survival by extracellular interaction and inactivation of the growth factor IGF-I. Beyond that, IGF-independent actions mediated by intracellular IGFBP-3 including nuclear-IGFBP-3, have also been described. We here show, using both confocal and electron microscopy and cell fractionation, that the extracellular addition of IGFBP-3 to living cells results in rapid uptake and nuclear delivery of IGFBP-3, by yet partly unknown mechanisms. IGFBP-3 is internalized through a dynamin-dependent pathway, traffics through endocytic compartments and is finally delivered into the nucleus. We observed docking of IGFBP-3 containing structures to the nuclear envelope and found IGFBP-3 containing dot-like structures to permeate the nuclear envelope. In summary, our findings establish the pathway by which this tumor suppressor protein is delivered from extracellular space to the nucleus.

Detection of human papillomavirus type 18 E7 oncoprotein in cervical smears: a feasibility study.

Persistent infections by high-risk human papillomaviruses (HPVs) are the main etiological factor for cervical cancer, and expression of HPV E7 oncoproteins was suggested to be a potential marker for tumor progression. The objective of this study was to generate new reagents for the detection of the HPV18 E7 oncoprotein in cervical smears. Rabbit monoclonal antibodies against recombinant E7 protein of HPV type 18 (HPV18) were generated and characterized using Western blotting, epitope mapping, indirect immunofluorescence, and immunohistochemistry. One clone specifically recognizing HPV18 E7 was used for the development of a sandwich enzyme-linked immunosorbent assay (ELISA). The assay was validated using recombinant E7 proteins of various HPV types and lysates from E7-positive cervical carcinoma cells. A total of 14 HPV18 DNA-positive cervical swab specimens and 24 HPV DNA-negative-control specimens were used for the determination of E7 protein levels by the newly established sandwich ELISA. On the basis of the average absorbance values obtained from all 24 negative controls, a cutoff above which a clinical sample can be judged E7 positive was established. Significant E7 signals 6- to 30-fold over background were found in 7 out of 14 abnormal HPV18 DNA-positive cervical smear specimens. This feasibility study demonstrates for the first time that HPV18 E7 oncoprotein can be detected in cervical smears.

Identification of human fumarylacetoacetate hydrolase domain-containing protein 1 (FAHD1) as a novel mitochondrial acylpyruvase.

The human fumarylacetoacetate hydrolase (FAH) domain-containing protein 1 (FAHD1) is part of the FAH protein superfamily, but its enzymatic function is unknown. In the quest for a putative enzymatic function of FAHD1, we found that FAHD1 exhibits acylpyruvase activity, demonstrated by the hydrolysis of acetylpyruvate and fumarylpyruvate in vitro, whereas several structurally related compounds were not hydrolyzed as efficiently. Conserved amino acids Asp-102 and Arg-106 of FAHD1 were found important for its catalytic activity, and Mg(2+) was required for maximal enzyme activity. FAHD1 was found expressed in all tested murine tissues, with highest expression in liver and kidney. FAHD1 was also found in several human cell lines, where it localized to mitochondria. In summary, the current work identified mammalian FAHD1 as a novel mitochondrial enzyme with acylpyruvate hydrolase activity.

Purification and characterization of native human insulin-like growth factor binding protein-6.

Insulin-like growth factor binding proteins (IGFBPs) are key regulators of insulin-like growth factor (IGF) mediated signal transduction and thereby can profoundly influence cellular phenotypes and cell fate. Whereas IGFBPs are extracellular proteins, intracellular activities were described for several IGFBP family members, such as IGFBP-3, which can be reinternalized by endocytosis and reaches the nucleus through routes that remain to be fully established. Within the family of IGFBPs, IGFBP-6 is unique for its specific binding to IGF-II. IGFBP-6 was described to possess additional IGF-independent activities, which have in part been attributed to its translocation to the nucleus; however, cellular uptake of IGFBP-6 was not described. To further explore IGFBP-6 functions, we developed a new method for the purification of native human IGFBP-6 from cell culture supernatants, involving a four-step affinity purification procedure, which yields highly enriched IGFBP-6. Whereas protein purified in this way retained the capacity to interact with IGF-II and modulate IGF-dependent signal transduction, our data suggest that, unlike IGFBP-3, human IGFBP-6 is not readily internalized by human tumor cells. To summarize, this work describes a novel and efficient method for the purification of native human insulin-like growth factor binding protein 6 (IGFBP-6) from human cell culture supernatants, applying a four-step chromatography procedure. Intactness of purified IGFBP-6 was confirmed by IGF ligand Western blot and ability to modulate IGF-dependent signal transduction. Cellular uptake studies were performed to further characterize the purified protein, showing no short-term uptake of IGFBP-6, in contrast to IGFBP-3.

Subcellular localization of the human papillomavirus 16 E7 oncoprotein in CaSki cells and its detection in cervical adenocarcinoma and adenocarcinoma in situ.

E7 is the major oncoprotein of high-risk human papillomaviruses (HPV) which causes cervical cancer. To date E7 oncoproteins have not been investigated in cervical adenocarcinoma. In this study we generated a rabbit monoclonal anti-HPV-16 E7 antibody, RabMab42-3, which recognizes a conformational epitope in the E7 carboxy-terminal zinc-finger resulting in a strong increase in the sensitivity for the detection of cell-associated HPV-16 E7 protein relative to conventional polyclonal anti-HPV-16 E7 antibodies. Using RabMab42-3, we show that the subcellular localization of endogenous HPV-16 E7 oncoprotein varies during the cell cycle in cervical cancer cells. Moreover, we demonstrate for the first time that the HPV-16 E7 oncoprotein is abundantly expressed in cervical adenocarcinoma in situ and adenocarcinoma, suggesting an important role of HPV-16 E7 for the development of these tumors. Our findings suggest that the HPV-16 E7 oncoprotein could be a useful marker for the detection of cervical adenocarcinoma and their precursors.

A new method for the purification of bioactive insulin-like growth factor-binding protein-3.

We present a novel efficient procedure for high level purification of human IGFBP-3. Insulin-like growth factor-binding proteins (IGFBPs) are key regulators of insulin-like growth factor mediated signal transduction and thereby can profoundly influence cellular phenotypes. Certain IGFBPs, including IGFBP-3, have also been described to possess additional IGF-independent activities, which rely, at least in part, on their nuclear localization. However, the mechanisms of IGF-independent biological activities of IGFBP-3 are not well understood. For the study of these functions, recombinant IGFBP-3 is used. However, it has traditionally been difficult to obtain recombinant protein in sufficient quality and quantity. Here we describe a new procedure for the purification of recombinant IGFBP-3 from cell culture supernatants involving a two-step affinity purification procedure. Using this new protocol, we obtained pure IGFBP-3 free of any visible contaminants. We also provide evidence that the protein purified in this way retains biological activity, to bind IGF and modulate IGF-dependent signal transduction. We also show that the purified protein produced by the new procedure is readily internalized by human fibroblasts, suggesting that this protein is also suitable to study intracellular trafficking of IGFBP-3.

The NADPH oxidase Nox4 restricts the replicative lifespan of human endothelial cells.

The free radical theory of aging proposes that ROS (reactive oxygen species) are major driving forces of aging, and are also critically involved in cellular senescence. Besides the mitochondrial respiratory chain, alternative sources of ROS have been described that might contribute to cellular senescence. Noxs (NADPH oxidases) are well-known sources of superoxide, which contribute to the antimicrobial capabilities of macrophages, a process involving the prototypical member of the family referred to as Nox2. However, in recent years non-phagocytic homologues of Nox2 have been identified that are involved in processes other than the host defence. Superoxide anions produced by these enzymes are believed to play a major role in signalling by MAPKs (mitogen-activated protein kinases) and stress-activated kinases, but could also contribute to cellular senescence, which is known to involve oxygen radicals. In HUVECs (human umbilical vein endothelial cells), Nox4 is predominantly expressed, but its role in replicative senescence of HUVECs remains to be elucidated. Using shRNA (small-hairpin RNA)-mediated knockdown of Nox4, implicating lentiviral vectors, we addressed the question of whether lifelong depletion of Nox4 in HUVECs would influence the senescent phenotype. We found a significant extension of the replicative lifespan of HUVECs upon knockdown of Nox4. Surprisingly, mean telomere length was significantly reduced in Nox4-depleted cells. Nox4 depletion had no discernable influence on the activity of MAPKs and stress-activated kinases, but reduced the degree of oxidative DNA damage. These results suggest that Nox4 activity increases oxidative damage in HUVECs, leading to loss of replicative potential, which is at least partly independent of telomere attrition.

Novel mechanism of IGF-binding protein-3 action on prostate cancer cells: inhibition of proliferation, adhesion, and motility.

IGF-binding protein-3 (IGFBP-3) is a modulator of the IGF-signaling pathway and was described as an anti-cancer agent in prostate cancer. The molecular mechanisms underlying these effects remained, however, largely undefined. We analyzed the influence of recombinant IGFBP-3 on cell proliferation of PC3, Du145, and LNCaP prostate cancer cells. As expected, IGFBP-3 inhibited IGF-stimulated cell proliferation by blocking IGF-mediated proliferation signals, but we observed an IGF-independent inhibitory effect of IGFBP-3 on prostate cancer cell proliferation in long-term cultures. We further investigated the influence of IGFBP-3 on adhesion, motility, and invasion of prostate cancer cells using adhesion assays, live-cell imaging techniques, and matrigel invasion measurements. There was a clear inhibitory effect of IGFBP-3 on tumor cell adhesion to extracellular matrix components in the presence and absence of IGF, whereas cell-cell adhesion was not affected. The same inhibitory effect of IGFBP-3 was determined on cell motility when real-time cell movements were followed. In addition, IGFBP-3 was able to inhibit tumor cell invasion through matrigel. In summary, we show that IGFBP-3 inhibits proliferation, adhesion, migration, and invasion processes of prostate tumor cells. These newly described mechanisms of IGFBP-3 can be of importance for tumor progression and support a role of IGFBP-3 in therapeutic settings.