Rhodamine-marked bombesin: a novel means for prostate cancer fluorescence imaging.

The gastrin releasing peptide receptor (GRPR) has been found to be strongly expressed in various types of cancers such as prostate and breast carcinomas. The GRPR ligands gastrin releasing peptide and bombesin can play a very significant role in cancer therapy and diagnostics. In this study we synthesized unlabeled bombesin BBN along with two conjugates in which the correct bombesin (BBN-Rhd) and a mutant bombesin (mBBN-Rhd) sequence was coupled to rhodamine, a fluorescent dye. These novel rhodamine fluorescent conjugates were used to study the targeting and uptake of bombesin on a cellular level. Nine different human cell lines including both tumor and healthy cells were examined using flow cytometry and confocal laser scanning microscopy. GRPR mRNA expression analysis was performed and it was found that the receptor is highly expressed in LNCaP and PC3 cells compared to the rest of other cell lines. Competition experiments were performed to verify the receptor dependence of the labeled conjugates using unmarked bombesin. The present study is a first attempt at direct fluorescence imaging of living cells using bombesin and its target, the GRPR. A rhodamine bombesin conjugate can be used as marker to differentiate between healthy cells and malignant cells such as prostate hyperplasia and prostate carcinoma in the early detection of cancer.

Novel bourgeonal fragrance conjugates for the detection of prostate cancer.

The methods used for detection of prostate cancer and prostate cancer lymph node metastases in medical diagnostics leave room for improvement. Currently, no means of identifying metastasized lymph nodes other than biopsies is available. Markers which are exclusively found on prostate cancer cells present a focal point for potential imaging methods. To complement the established markers like e.g. PCA3-a noncoding mRNA sequence-and PSA-a serine protease-we investigated the ectopically expressed G-protein coupled olfactory receptor OR1D2 as a possible target for prostate-specific detection with its agonist bourgeonal which has been conjugated to two different fluorescent dyes. We performed mRNA expression analysis of the OR1D2 receptor mRNA by reverse transcriptase polymerase chain reaction on LNCaP prostate carcinoma cells and three other non-prostate derived carcinoma cell lines. Additionally, we used flow cytometry to investigate the uptake of fluorescent-dye-bound OR1D2-ligand bourgeonal into the examined carcinoma cell lines. Finally, confocal laser scanning microscopy of in vitro cell culture and in vivo tumor xenografts on mice was performed. We could confirm OR1D2 receptor mRNA overexpression as well as stronger uptake of both bourgeonal conjugates in vitro and in vivo for LNCaP cells compared to the non-prostate derived cell lines. Cytoplasmic accumulation and no adverse effects after in vitro and in vivo application of the conjugates were observed. The conjugates represent a platform for the development of future prostate-specific imaging applications, e.g. detection of metastasized lymph nodes during surgery by intraoperative laser examination.

Using the neurotransmitter serotonin to target imaging agents to glioblastoma cells.

The neurotransmitter serotonin is involved in numerous bodily functions via seven different serotonin receptor subfamilies. Serotonin plays a role in gastrointestinal functions like intestinal secretion or peristalsis and neuropsychiatric events like depression or migraine. One of these subtypes has been found on glioblastoma cells, inducing growth promotion. In our study we attempted to target imaging agents to glioblastoma cells via the serotonin receptor. For this we coupled serotonin to the fluorescent dye rhodamine and the magnetic resonance imaging contrast agent gadolinium (Gd)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). The cellular uptake, cytotoxicity and detection sensitivity of the conjugates were evaluated by confocal laser scanning microscopy (CLSM), cell growth analysis, flow cytometry and magnetic resonance relaxometry on U373 human glioblastoma cells. Receptor-dependency of the uptake was confirmed by competition experiments with excess of unmarked serotonin. Cellular uptake of the conjugates was found in CLSM, magnetic resonance relaxometry and flow cytometry experiments.CLSM revealed the cytoplasmic character of the uptake. In cell growth analysis experiments no adverse effect of either conjugate on the cells was observed. Competition experiments performed with the conjugates and unmarked serotonin showed decreased conjugate uptake compared to the experiments without competition. In conclusion the neurotransmitter serotonin could be successfully used to target imaging agents into human glioblastoma cells. This makes it of interest for future glioblastoma imaging methods.

Dermcidin-derived peptides show a different mode of action than the cathelicidin LL-37 against Staphylococcus aureus.

Dermcidin (DCD) is an antimicrobial peptide which is constitutively expressed in eccrine sweat glands. By postsecretory proteolytic processing in sweat, the DCD protein gives rise to anionic and cationic DCD peptides with a broad spectrum of antimicrobial activity. Many antimicrobial peptides induce membrane permeabilization as part of their killing mechanism, which is accompanied by a loss of the bacterial membrane potential. In this study we show that there is a time-dependent bactericidal activity of anionic and cationic DCD-derived peptides which is followed by bacterial membrane depolarization. However, DCD-derived peptides do not induce pore formation in the membranes of gram-negative and gram-positive bacteria. This is in contrast to the mode of action of the cathelicidin LL-37. Interestingly, LL-37 as well as DCD-derived peptides inhibit bacterial macromolecular synthesis, especially RNA and protein synthesis, without binding to microbial DNA or RNA. Binding studies with components of the cell envelope of gram-positive and gram-negative bacteria and with model membranes indicated that DCD-derived peptides bind to the bacterial envelope but show only a weak binding to lipopolysaccharide (LPS) from gram-negative bacteria or to peptidoglycan, lipoteichoic acid, and wall teichoic acid, isolated from Staphylococcus aureus. In contrast, LL-37 binds strongly in a dose-dependent fashion to these components. Altogether, these data indicate that the mode of action of DCD-derived peptides is different from that of the cathelicidin LL-37 and that components of the bacterial cell envelope play a role in the antimicrobial activity of DCD.

Chemosensitization of Prostate Carcinoma Cells with a Receptor-directed Smac Conjugate.

BACKGROUND: Second mitochondrial activator of caspase (Smac) is a short mitochondrial peptide. When released from the mitochondria into the cytoplasm, it binds to inhibitor of apoptotic proteins (IAPs) within the cytoplasm and prevents them from inhibiting apoptosis. OBJECTIVE: Delivery of external synthetic Smac peptide into the cytoplasm of malignant cells could greatly improve the efficiency of apoptosis-inducing chemotherapeutic agents. METHOD: In our study different conjugates based on the seven N-terminal amino acids AVPIAQK of Smac (SmacN7) were produced to obtain a cytoplasm-directed Smac variant. SmacN7 and a point mutant (AVPKAQK) were coupled either to rhodamine alone or to both rhodamine and undecylic aldehyde, which is an antagonist of the Lily-of-the-valley fragrance receptor. The fifth conjugate consisted of rhodamine coupled only to undecylic aldehyde, without SmacN7. The uptake of these five conjugates into three different human cell lines was characterized and quantified by confocal laser scanning microscopy and flow cytometry. A caspase apoptosis assay was performed for cells incubated with the five different conjugates after induction of apoptosis. RESULTS: The coupling of undecylic aldehyde to SmacN7 increased the cellular uptake of the correct and mutant conjugates. CONCLUSION: Caspase 3/7 apoptosis tests after induction of apoptosis with staurosporine or UV irradiation showed that the coupling of SmacN7 with undecylic aldehyde resulted in a greatly increased adjuvant pro-apoptotic effect compared to the separate components and a mutant SmacN7 peptide sequence in the LNCaP prostate carcinoma cells compared to the benign prostate hyperplasia (BPH) cells and the human embryonal kidney (HEK) cells.

Detection of dermcidin-derived peptides in sweat by ProteinChip technology.

Recently, a novel antimicrobial peptide DCD-1, derived from the Dermcidin (DCD) gene and secreted by sweat glands, has been described by Schittek et al. [Nat. Immunol. 2 (2001) 1133.]. Here we describe the application of the surface-enhanced laser desorption/ionisation (SELDI) technology for the detection of DCD-1 and other dermcidin-derived peptides directly from microlitre amounts of human sweat. The advantages of the technique are as follows: (a) it can be carried out with ease and rapidity; (b) multiple samples can be processed simultaneously; (c) prior purification is not required; and (d) only a limited sample volume is necessary for both protein profiling and semiquantitation. Profiling of human sweat from various donors revealed that in addition to DCD-1, other DCD-derived peptide species were also present in significant quantities. Four of five identified peptides were DCD-1 related, while the fifth corresponded to a portion of the DCD protein outside the DCD-1 core. This provides clues as to how the novel protein is processed to its active form, though further work remains to elucidate this fully. Thus, we have demonstrated the applicability of such technology to the detection of DCD-1 and for the protein profiling of sweat in general. Such studies could reveal valuable new biomarkers for diagnosis and treatment of skin and sweat gland disorders.

Identification of an in vitro insulin receptor substrate-1 phosphorylation site by negative-ion muLC/ES-API-CID-MS hybrid scan technique.

Recently, we reported a fast on-line alkaline micro-liquid chromatography/electrospray-atmospheric pressure ionization/collision-induced dissociation/mass spectrometric approach for sensitive phosphopeptide screening of a tryptic digested protein and subsequent characterization of the identified phosphopeptide. Based on this study, we now applied an improved method for the identification of phosphorylation sites in insulin receptor substrate 1, an important mediator in insulin signal transduction which was phosphorylated in vitro by protein kinase C-zeta. The approach consists of an on-line alkaline negative-ion micro-liquid chromatography/electrospray-atmospheric pressure ionization/collision-induced dissociation/mass spectrometric hybrid scan experiment using a triple-quadrupole mass spectrometer with fractionation and subsequent off-line nanoES-MS (ion trap) analysis of the phosphopeptide-containing fractions. During the liquid chromatography (LC)/ES-MS experiment, the phosphopeptides of the enzymatic digest mixture of the studied insulin receptor substrate 1 fragment were detected under high skimmer potential (API-CID) using phosphorylation-specific m/z 79 marker ions as well as the intact m/z-values of the peptides which were recorded under low skimmer potential. Subsequently, the targeted fractions were analyzed by off-line nanoES-MS/MS and MS(3). Using this approach, serine 318 was clearly identified as a major in vitro protein kinase C-zeta phosphorylation site in the insulin receptor substrate -1 fragment. Together, our results indicate that the applied strategy is useful for unequivocal and fast analysis of phosphorylation sites in low abundant signaling proteins.

Th2 dominance of T helper cell response to preproinsulin in individuals with preclinical type 1 diabetes.

In human type 1 diabetes (T1D) autoantibodies to insulin precede clinical disease, while little is known about the contribution of insulin-specific T lymphocytes-in particular, T helper (Th) subsets. Here we have studied the in vivo primed cytokine response to preproinsulin in peripheral blood mononuclear cells (PBMCs) and two major Th cell subsets-CD45RO+ memory cells and CD45RA+ naive/resting cells-in 35 individuals with HLA-DRB1*04, DQB1*0302 diabetes risk marker: 12 patients with T1D, 12 autoantibody-positive (Ab+) individuals, and 11 healthy controls. Cytokine secretion (TNF-alpha, IFN-gamma, IL-2, IL-4, IL-5, and IL-10) was measured in the supernatants of the cultures stimulated with 21 overlapping preproinsulin peptides as well as proinsulin and insulin. In Ab+ individuals our results reveal higher IL-4 levels in CD45RO+ memory cells and higher IL-5 levels in CD45RA+ naive/resting cells, while higher IL-2 production was found in PBMCs. In contrast, in PBMCs of T1D patients higher IFN-gamma and IL-10 secretion was found. Our data delineate characteristic cytokine patterns in peripheral T lymphocytes from patients at different stages of the T1D development.

Relationship between T and B cell responses to proinsulin in human type 1 diabetes.

In type 1 diabetes, humoral and cell-mediated responses to insulin and proinsulin are detectable. Autoantibodies to insulin are associated with impending disease in young individuals and are used as predictive markers to determine disease risk. The aim of this study was to investigate whether different cytokine patterns of cellular reactivity to insulin might serve as additional specific markers of disease maturation and might improve disease prediction in individuals at risk. We correlated T and B cell responses to insulin in subjects with increased genetic risk (HLA-DRB1*04, DQB1*0302) for diabetes with or without islet autoantibodies (Ab+ subjects and controls, respectively) and HLA-matched patients. Peripheral blood mononuclear cells were stimulated with 15 overlapping proinsulin peptides (16-mer), and proinflammatory Th1 (IFNgamma) and anti-inflammatory Th2 (IL-4) cytokines were analyzed. We observed a simultaneous increase in IL-4 and IFNgamma secretion in early islet autoimmunity of Ab+ subjects, but not in insulin-treated T1D patients. Furthermore, the increase in IL-4 secretion in Ab+ subjects was associated with insulin autoantibody responses. There was no correlation of either IFNgamma or IL-4 secretion with insulin antibody responses in patients already treated with exogenous insulin. In conclusion, our findings reveal that quantification of cytokine responses to proinsulin in peripheral blood may prove to be a promising specific marker of diabetes progression and could, in addition to insulin autoantibodies, be used in the prediction of type 1 diabetes.

Ramoplanin imaging conjugates--synthesis and evaluation.

In a previous study we found that fluorescence-marked vancomycin--a glycopeptide antibiotic--is taken up into human tumor cells. To expand on these investigations we now used the lipoglycodepsipeptide antibiotic ramoplanin. Compared to vancomycin it is not only a bigger molecule, but it also has two potential binding sites for coupling to the imaging agents. Three different ramoplanin imaging conjugates were synthesized, two used for fluorescence imaging and one for magnetic resonance imaging. The two fluorescent dyes used in confocal laser scanning microscopy (CLSM) and fluorescence activated cell sorting (FACS) were fluorescein isothiocyanate (FITC) and rhodamine isothiocyanate (RITC). The third was the magnetic resonance imaging (MRI) contrast agent gadolinium-1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid (GdDOTA). The uptake of ramoplanin conjugates, their specificity for different cell lines and the accessibility of the conjugates by imaging methods were evaluated on 8 human cell lines (two benign, six malignant) by CLSM, FACS and MRI experiments. Cytotoxicity of the ramoplanin conjugates was determined in the FACS experiments with the propidium iodide and Annexin-V-Fluos indicating any disruption in the cell membranes. Cytoplasmic uptake of the ramoplanin conjugates was observed in confocal laser scanning images and was measured using FACS and MRI experiments. Compared to the vancomycin conjugates the ramoplanin conjugates showed much weaker and slower uptake. Additionally, uptake of the ramoplanin conjugates led to strong membrane disruption and cell death.

The gastrin/cholecystokinin-B receptor on prostate cells--a novel target for bifunctional prostate cancer imaging.

The means of identifying prostate carcinoma and its metastases are limited. The contrast agents used in magnetic resonance imaging clinical diagnostics are not taken up into the tumor cells, but only accumulate in the interstitial space of the highly vasculated tumor. We examined the gastrin/cholecystokinin-B receptor as a possible target for prostate-specific detection using the C-terminal seven amino acid sequence of the gastrin peptide hormone. The correct sequence and a scrambled control sequence were coupled to the fluorescent dye rhodamine and the magnetic resonance imaging contrast agent gadolinium (Gd)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). Expression analysis of the gastrin receptor mRNA was performed by reverse transcriptase polymerase chain reaction on PC3 prostate carcinoma cells, U373 glioma, U2OS osteosarcoma and Colo205 colon carcinoma cells. After having confirmed elevated expression of gastrin receptor in PC3 cells and very low expression of the receptor in Colo205 cells, these two cell lines were used to create tumor xenografts on nude mice for in vivo experiments. Confocal lasers scanning microscopy and magnetic resonance imaging showed a high specificity of the correct conjugate for the PC3 xenografts. Staining of the PC3 xenografts was much weaker with the scrambled conjugate while the Colo205 xenografts showed no marked staining with any of the conjugates. In vitro experiments comparing the correct and scrambled conjugates on PC3 cells by magnetic resonance relaxometry and fluorescence-activated cell sorting confirmed markedly higher specificity of the correct conjugate. The investigations show that the gastrin receptor is a promising tumor cell surface target for future prostate-cancer-specific imaging applications.

Novel gastrin receptor-directed contrast agents - potential in brain tumor magnetic resonance imaging.

Magnetic resonance imaging (MRI) is presently the method of choice for detection of brain tumors. However, MRI alone is not conclusive. As the commonly used contrast agents do not bind to the cells and are not taken up into the cells, they generally do accumulate in regions where the blood-brain-barrier is disrupted. While this can be brain tumors (WHO grade II-III and above), it can also be inflammations. A cell-directed contrast agent would be a great asset not only to avoid unnecessary brain biopsies, but also to achieve sharper tumor margins during intraoperative MRI. The gastrin/cholecystockinin receptor found in the brain and the intestinal tract is a potential target for a cell-directed contrast agent. The receptor has already been found in human glioma cell lines and autocrine stimulation has also been demonstrated for the receptor and its ligand gastrin. We coupled the correct and a mutant 17-amino-acid gastrin to gadolinium -1,4,7,10-tetraazacyclododecane-1,4,7,10- tetraacetic acid (an MRI contrast agent) and rhodamine isothiocyanate (a fluorescent dye). Using confocal laser scanning microscopy and magnetic resonance relaxometry experiments we found cytoplasmic uptake of the correct gastrin conjugate into human U373 glioma cells. Surprisingly, the mutant conjugate was also taken up into the cells in a similar pattern, albeit to a lesser degree. Both conjugates showed no cytotoxicity. These conjugates show potential for future use in magnetic resonance imaging studies of brain tumors after systemic or intraoperative local application. The cytoplasm specificity of the conjugates also makes it a potential building block for the design of future cytoplasmdirected imaging and therapeutic conjugates.

Potential of the gastric motility drug lorglumide in prostate cancer imaging.

The use of tissue-specific receptor ligands is a promising approach for cancer diagnostics and therapy. Lorglumide, a highly effective competitive ligand for the cholecystokinine-A receptor (CCKRA) was conjugated to a fluorescent dye and a magnetic resonance imaging (MRI) contrast agent to obtain a bifunctional marker for tissue with high CCKRA expression. An intermediate conjugate containing only lorglumide and a fluorescent dye was also produced. By performing CCKRA mRNA expression analysis on carcinoma cell lines we found that CCKRA is highly expressed in PC3 prostate carcinoma cells compared to U373 glioma and U2OS osteosarcoma cells. Uptake, specificity and detection sensitivity of both lorglumide conjugates was evaluated by confocal laser scanning microscopy, fluorescence activated cell sorting (FACS) and magnetic resonance relaxometry. While the conjugate containing only lorglumide and rhodamine isothiocyanate as fluorescent dye showed clearly higher uptake than the bifunctional conjugate in FACS analysis, both conjugates clearly showed preferential staining of the PC3 prostate carcinoma cells. Magnetic resonance relaxometry experiments with the bifunctional conjugate containing the MRI contrast agent gadolinium-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid confirmed the higher PC3-affinity of the lorglumide ligand. Confocal laser scanning microscopy images of PC3/U2OS mixed cell cultures incubated with the bifunctional conjugate also clearly showed PC3 preference and cytoplasmic dot-like staining concurring with uptake by receptor binding and subsequent receptor internalization. Considering these results, CCKRA ligands like lorglumide could play a role in the future design of prostate-cancer-specific markers.

Evaluating the diagnostic and chemotherapeutic potential of vancomycin-derived imaging conjugates.

The main use of glycopeptide antibiotics is treatment of infections which are resistant to the commonly used ß- lactam antibiotics. Antitumor activity has also been reported for some glycopeptide antibiotics like bleomycin. In the present study we investigated the chemotherapeutic and diagnostic potential of two imaging agent derivatives of the glycopeptide antibiotic vancomycin. For the first conjugate, vancomycin was coupled to the fluorescent dye rhodamine, used in confocal laser scanning microscopy (CLSM) and fluorescence-activated cell sorting (FACS). The second conjugate consisted of vancomycin coupled to gadolinium-1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid (GdDOTA), a magnetic resonance imaging (MRI) contrast agent. The cellular uptake, specificity, and the accessibility by imaging methods of the two vancomycin conjugates was evaluated on 8 human cell lines (one benign, 7 malignant) by CLSM, FACS, and MRI experiments. Cytotoxicity of both vancomycin conjugates was determined in the FACS experiments with the annexin test indicating disrupted cell membranes. Some of the malignant cell lines showed clearly stronger uptake than the others and the benign cell line was among the cell lines with the lowest uptake. In the annexin test the cytotoxicity could be correlated to the conjugate uptake for all cell lines. The intracellular uptake of the vancomycin conjugates and the increased uptake into some of the malignant cell lines were interesting findings which should be further pursued.

Peptides derived from the human laminin alpha4 and alpha5 chains exhibit antimicrobial activity.

Laminins are a family of heterotrimeric extracellular matrix glycoproteins in the basement membrane of different tissues and are composed of alpha, beta, and gamma chains. In mammals, five different alpha chains, three beta chains, and three gamma chains have been identified that assemble into 15 different laminins. Each alpha-chain possesses a C-terminal globular domain which can be subdivided into the five subdomains LG1-LG5. LG1-LG3 modules are connected to LG4-LG5 by a linker domain which is known to be sensitive to proteolytic processing. Here, we show that peptides derived from the human laminin alpha4 and alpha5 chain, exhibit a dose-dependent antimicrobial activity against gram-positive and gram-negative bacteria. Furthermore, we show that these peptides permeabilize the bacterial membrane and are able to bind to bacterial DNA. Interestingly, the ability to kill the microorganisms correlated with their ability to bind to heparin. These data suggest that extracellular matrix components are able to protect the respective tissues from invading pathogens and are part of the host defense response.

Serum protein profiling by SELDI mass spectrometry: detection of multiple variants of serum amyloid alpha in renal cancer patients.

The molecular analysis of serum is an important field for the definition of potential diagnostic markers or disease-related protein alterations. Novel proteomic technologies such as the mass spectrometric-based surface-enhanced laser desorption/ionization (SELDI) ProteinChip technique facilitate a rapid and reproducible analysis of such protein mixtures and affords the researcher a new dimension in the search for biomarkers of disease. Here, we have applied this technology to the study of a cohort of serum samples from well-characterized renal cell carcinoma patients for the identification of such proteins by comparison to healthy controls. We detected and characterized haptoglobin 1 alpha and serum amyloid alpha-1 (SAA-1) as disease related, in addition to an as-yet-unidentified marker of 10.84 kDa. Of particular note is the detection of multiple variants of SAA-1 in multiplex that have not been described in the sera of cancer patients. SAA-1 is detected as full-length protein, des-Arginine and des-Arginine/des-Serine variants at the N terminus by SELDI. In addition, we could also detect a low-abundant variant minus the first five N-terminal amino acids. Such variants may impact the function of the protein. We conclude the technique to be a reproducible, fast and simple mode for the discovery and analysis of marker proteins of disease in serum.

Human alpha-defensins HNPs-1, -2, and -3 in renal cell carcinoma: influences on tumor cell proliferation.

The alpha-defensins human neutrophil peptides (HNPs)-1, -2, and -3 have been described as cytotoxic peptides with restricted expression in neutrophils and in some lymphocytes. In this study we report that HNPs-1, -2, and -3 are also expressed in renal cell carcinomas (RCCs). Several RCC lines were found to express mRNA as well as the specific peptides of HNP-1, -2, and -3 demonstrated by reverse transcriptase-polymerase chain reaction, mass spectrometric, and flow cytometric analyses. At physiological concentrations HNPs-1, -2, and -3 stimulated cell proliferation of selected RCC lines in vitro but at high concentrations were cytotoxic for all RCC lines tested. As in RCC lines, alpha-defensins were also detected in vivo in malignant epithelial cells of 31 RCC tissues in addition to their expected presence in neutrophils. In most RCC cases randomly, patchy immunostaining of alpha-defensins on epithelial cells surrounding neutrophils was seen, but in six tumors of higher grade malignancy all tumor cells were diffusely stained. Cellular necrosis observed in RCC tissues in association with extensive patches of HNP-1, -2, and -3, seemed to be related to high concentrations of alpha-defensins. The in vitro and in vivo findings suggest that alpha-defensins are frequent peptide constituents of malignant epithelial cells in RCC with a possible direct influence on tumor proliferation.