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A phase Ib/II trial was performed to evaluate safety, tolerability, recommended dose (RD) and efficacy of F16-IL2, a recombinant antibody-cytokine fusion protein, in combination with doxorubicin in patients with solid tumors (phase Ib) and metastatic breast cancer (phase II). Six patient cohorts with progressive solid tumors (n = 19) received escalating doses of F16-IL2 [5–25 Million International Units (MIU) of IL2 equivalent dose] in combination with escalating doses of doxorubicin (0–25 mg/m2) on day 1, 8 and 15 every 4 weeks. Subsequently, patients with metastatic breast cancer (n = 10) received the drug combination at the RD. Clinical data and laboratory findings were analyzed for safety, tolerability, and activity. F16-IL2 could be administered up to 25 MIU, in combination with the RD of doxorubicin (25 mg/m2). No human anti-fusion protein antibodies (HAFA) response was detected. Pharmacokinetics of F16-IL2 was dose-dependent over the tested range, with half-lives of ca. 13 and ca. 8 hours for cohorts dosed at lower and higher levels, respectively. Toxicities were controllable and reversible, with no combination treatment-related death. After 8 weeks, 57% and 67% disease control rates were observed for Phase I and II, respectively (decreasing to 43% and 33% after 12 weeks), considering 14 and 9 patients evaluable for efficacy. One patient experienced a long lasting partial response (45 weeks), still on-going at exit of study. F16-IL2 can be safely and repeatedly administered at the RD of 25 MIU in combination with 25 mg/m2doxorubicin; its safety and activity are currently being investigated in combination with other chemotherapeutics, in order to establish optimal therapy settings.

Tissue inhibitor of metalloproteinases-1 (TIMP-1) inhibits matrix metalloproteinases (MMPs) by binding at a 1:1 stoichiometry. Here we have shown the involvement of N-glycosylation in the MMP inhibitory ability of TIMP-1. TIMP-1, purified from HEK 293 cells overexpressing TIMP-1 (293 TIMP-1), showed less binding and inhibitory abilities to MMPs than TIMP-1 purified from fibroblasts or SF9 insect cells infected with TIMP-1 baculovirus. Following deglycosylation of TIMP-1, all forms of TIMP-1 showed similar levels of MMP binding and inhibition, suggesting that glycosylation is involved in the regulation of these TIMP-1 activities. Analysis of the N-glycan structures showed that SF9 TIMP-1 has the simplest N-glycan structures, followed by fibroblast TIMP-1 and 293 TIMP-1, in order of increasing complexity in their N-glycan structures. Further analyses showed that cleavage of outer arm fucose residues from the N-glycans of 293 TIMP-1 or knockdown of both FUT4 and FUT7 (which encode for fucosyltransferases that add outer arm fucose residues to N-glycans) enhanced the MMP-binding and catalytic abilities of 293 TIMP-1, bringing them up to the levels of the other TIMP-1. These results demonstrate that the ability of TIMP-1 to inhibit MMPs is at least in part regulated by outer arm fucosylation of its N-glycans.

Regulator of G-protein signaling 18 (RGS18) is a GTPase-activating protein for the G-α-q and G-α-i subunits of heterotrimeric G-proteins that turns off signaling by G-protein coupled receptors. RGS18 is highly expressed in platelets. In the present study, we show that the 14-3-3γ protein binds to phosphorylated serines 49 and 218 of RGS18. Platelet activation by thrombin, thromboxane A2, or ADP stimulates the association of 14-3-3 and RGS18, probably by increasing the phosphorylation of serine 49. In contrast, treatment of platelets with prostacyclin and nitric oxide, which trigger inhibitory cyclic nucleotide signaling involving cyclic AMP-dependent protein kinase A (PKA) and cyclic GMP-dependent protein kinase I (PKGI), induces the phosphorylation of serine 216 of RGS18 and the detachment of 14-3-3. Serine 216 phosphorylation is able to block 14-3-3 binding to RGS18 even in the presence of thrombin, thromboxane A2, or ADP. 14-3-3–deficient RGS18 is more active compared with 14-3-3–bound RGS18, leading to a more pronounced inhibition of thrombin-induced release of calcium ions from intracellular stores. Therefore, PKA- and PKGI-mediated detachment of 14-3-3 activates RGS18 to block Gq-dependent calcium signaling. These findings indicate cross-talk between platelet activation and inhibition pathways at the level of RGS18 and Gq.

Regulator of G-protein signaling 18 (RGS18) is a GTPase-activating protein for the G-α-q and G-α-i subunits of heterotrimeric G-proteins that turns off signaling by G-protein coupled receptors. RGS18 is highly expressed in platelets. In the present study, we show that the 14-3-3γ protein binds to phosphorylated serines 49 and 218 of RGS18. Platelet activation by thrombin, thromboxane A2, or ADP stimulates the association of 14-3-3 and RGS18, probably by increasing the phosphorylation of serine 49. In contrast, treatment of platelets with prostacyclin and nitric oxide, which trigger inhibitory cyclic nucleotide signaling involving cyclic AMP-dependent protein kinase A (PKA) and cyclic GMP-dependent protein kinase I (PKGI), induces the phosphorylation of serine 216 of RGS18 and the detachment of 14-3-3. Serine 216 phosphorylation is able to block 14-3-3 binding to RGS18 even in the presence of thrombin, thromboxane A2, or ADP. 14-3-3–deficient RGS18 is more active compared with 14-3-3–bound RGS18, leading to a more pronounced inhibition of thrombin-induced release of calcium ions from intracellular stores. Therefore, PKA- and PKGI-mediated detachment of 14-3-3 activates RGS18 to block Gq-dependent calcium signaling. These findings indicate cross-talk between platelet activation and inhibition pathways at the level of RGS18 and Gq.

The importance of the protein corona formed around nanoparticles upon entering a biological fluid has recently been highlighted. This corona is, when sufficiently long-lived, thought to govern the particles’ biological fate. However, even this long-lived “hard” corona evolves and re-equilibrates as particles pass from one biological fluid to another, and may be an important feature for long-term fate. Here we show the evolution of the protein corona as a result of transfer of nanoparticles from one biological fluid (plasma) into another (cytosolic fluid), a simple illustrative model for the uptake of nanoparticles into cells. While no direct comparison can be made to what would happen in, for example, the uptake pathway, the results confirm that significant evolution of the corona occurs in the second biological solution, but that the final corona contains a “fingerprint” of its history. This could be evolved to map the transport pathways utilized by nanoparticles, and eventually to predict nanoparticle fate and behavior.

We have analyzed the proteomes of two human melanoma cell lines (A375 and 526), and of the human melanocytes, (FOM 78), by two-dimensional electrophoresis (2D-PAGE) and liquid chromatography – tandem mass spectrometry (LC-MS/MS). Our comparative proteomic analysis revealed that six proteins were over-expressed in both melanoma cell lines as compared to melanocytes: galectin-1, inosine-5'-monophosphate dehydrogenase 2, serine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A alpha isoform, protein DJ-1, cyclophilin A and cofilin-1. We show, for the first time, that only specific isoforms of these molecules are over-expressed in melanoma. Different protein profiles were also found between each individual melanoma cell line and the melanocytes. s-Methyl-5-thioadenosine phosphorylase, ubiquitin and ribosomal protein S27 a precursor, the basic form of protein DJ-1, annexin a1, proliferation associated protein 2g4, isoform alfa-enolase of alfa-enolase, protein disulfide-isomerase precursor, and elongation factor 2 were more strongly expressed in A375 cells compared to melanocytes. In 526 cells, 60s acidic ribosomal protein p1 and calreticulin precursor were more highly expressed than in melanocytes. These molecular differences may help in better understanding melanoma development and its different responsiveness to therapies. The identified proteins could be exploited as biomarkers or therapeutic targets for melanoma.

A promising avenue toward the development of more selective anticancer drugs consists in the targeted delivery of bioactive molecules to the tumor environment by means of binding molecules specific to tumor-associated markers. We have used a chemical proteomics approach based on the ex vivoperfusion and biotinylation of accessible structures within surgically resected human kidneys with tumor to gain information about accessible and abundant antigens that are overexpressed in human cancer. This procedure led to the selective labeling with biotin of vascular structures. Biotinylated proteins were purified on streptavidin resin and identified using mass spectrometric methodologies, revealing 637 proteins, 184 of which were only found in tumor specimens and 223 of which were only found in portions of normal kidneys. Immunohistochemical and PCR analysis confirmed that several of the putative cancer antigens identified in this study are indeed preferentially expressed in tumors. In conclusion, we have developed a methodology that allows the identification of accessible biomarkers in human tissues. The tumor-associated antigens identified in this study may be suitable targets for antibody-based anticancer therapies. The experimental approach described here should be applicable to other surgical specimens and to other pathologies as well as to the study of basic physiological and immunological processes.

Angiogenesis, the sprouting of new blood vessels from preexisting ones, is a phenomenon associated to several human pathologies, including different potentially blinding ocular disorders, rheumatoid arthritis, and cancer. Indeed, ongoing angiogenesis is a characteristic feature of the majority of aggressive solid tumors, and is also a pre-requisite for the progression towards the metastatic phenotype. One established marker of angiogenesis is represented by an isoform of the oncofoetal fibronectin (FN), containing an additional domain inserted by alternative splicing of the FN pre-mRNA and called extra-domain B (ED-B). This isoform has been found to be present almost exclusively in the modified extra-cellular matrix surrounding newly-formed blood vessels in tumors (and other animal models of ocular pathologies), being completely absent from the normal vasculature in adult organs. This article reviews the recombinant antibodies raised against ED-B and the different methodologies used for the generation of these antibodies. Moreover, new diagnostic and therapeutic applications based on the delivery of bioactive molecules to tumor blood vessels by means of ED-B targeting will be discussed.

The cytoprotective role of heat shock proteins (HSP) described in variety of human diseases, including ischemia, inflammation, and infection, suggests new therapeutic strategies relying upon the development of drugs that selectively turn on heat shock genes. Cyclopentenone prostaglandins, which contain an α,β-unsaturated carbonyl group in the cyclopentane ring and possess antiviral activity against several RNA and DNA viruses, were shown to function as signal for HSP synthesis in a nonstressful situation in a variety of mammalian cells. We now report that 2-cyclopenten-1-one selectively induces the expression of the 70-kDa HSP (HSP70) in human cells, through cycloheximide-sensitive activation of heat shock transcription factor 1 (HSF1). The α,β-unsaturated carbonyl group is the key structure triggering HSF1 activation. Induction is associated with antiviral activity during infection with vesicular stomatitis virus. These results identify the molecular structure of natural prostaglandins responsible for HSF1 activation and open new perspectives in the search for novel antiviral and cytoprotective drugs.

We have used binding of radioactive lectins (i.e. Concanavalin A (ConA), Wheat Germ Agglutinin (WGA) andRicinus communisagglutinin I (RCAI)) to membrane glycoproteins separated in SDS gel electrophoresis, to detect specific carbohydrate changes in plasma membrane proteins ofin vivopassaged Friend erythroleukemia cells (FLC). These cells are highly metastatic to the liver, whereas the originalin vitropassaged tumor cells do not metastasize. Marked qualitative differences in the high molecular weight region of the gels (100–200 kD) were observed between the WGA binding glycoproteins of metastaticin vivopassaged FLC and nonmetastaticin vitropassaged FLC. Furthermore, the binding of WGA to plasma membrane proteins ofin vivopassaged FLC was much greater than the binding of WGA to plasma membrane proteins ofin vitropassaged FLC. Lectin binding experiments after sialic acid removal byin situmild acid hydrolysis of FLC glycoproteins indicated that an increased sialylation of the 120 and 145 kD glycoproteins was responsible for the increased WGA reactivity ofin vivopassaged FLC plasma membranes. Besides the increased sialylation, other changes in glycosylation of the 100–200 kD glycoproteins ofin vivopassaged FLC were observed: (1) qualitative differences between the WGA binding patterns of the two cell types were restored after treatment of the gels with mild acid and subsequent Smith degradation; (2) after chemical removal of sialic acid residues from the gels, qualitative differences in the RCA binding patterns to the glycoproteins of the two cell types were apparent.

Heat shock proteins (HSPs) are induced in mammalian cells in a variety of pathophysiological states and have an important role in cytoprotection in vitroand in vivo.In this study, we report that the calcium ionophore A23187, a glucose-regulated protein (GRP) inducer, dramatically inhibits HSP70 synthesis and HSP70mRNA transcription after induction by heat shock, sodium arsenite, or prostaglandin A1treatment in human K562 cells. A23187 does not suppress, and it actually prolongs, the DNA-binding activity of the human heat shock transcription factor (HSF), while it alters HSF1 phosphorylation in heat shock-treated cells. To inhibit HSP70 expression, A23187 needs to be present during heat shock, while treatment before or after heat shock does not affect HSP70mRNA transcription. The GRP inducer thapsigargin, which specifically inhibits the endoplasmic reticulum Ca2+-ATPase, has no effect on heat-induced HSP70 synthesis, indicating that A23187 inhibitory activity is not due to depletion of intracellular calcium stores and is independent of the concomitant induction of GRP genes. Inhibition of HSP70 expression is correlated with alterations in HSF1 phosphorylation in heat-shocked cells, but not in sodium arsenite-treated cells, indicating that different mechanisms may be involved in mediating A23187 inhibitory activity.

Since its discovery in the first half of the twentieth century, the high‐affinity, noncovalent interaction between biotin (vitamin H) and the avian protein avidin (and its bacterial homologs) has been exploited for many diverse biotechnology applications. This unit provides several basic protocols for labeling various protein reactive groups with biotin. These protocols can be applied not only to labeling in vitro or in tissue culture, but also to in vivo labeling of whole laboratory animals or to ex vivo labeling of surgically resected organs. Curr. Protoc. Protein Sci. 60:3.6.1‐3.6.21. © 2010 by John Wiley & Sons, Inc.