Synthesis of a Novel Curcumin Derivative as a Potential Imaging Probe in Alzheimer's Disease Imaging.

BACKGROUND: Curcumin has been of interest in the field of Alzheimer's disease. Early studies on transgenic mice showed promising results in the reduction of amyloid plaques.However, curcumin is very poorly soluble in aqueous solutions and not easily accessible to coupling as it contains only phenolic groups as potential coupling sites. For these reasons only few imaging studies using curcumin bound as an ester were performed and curcumin is mainly used as nutritional supplement. METHODS: In the present study we produced an aminoethyl ether derivative of curcumin using a nucleophilic substitution reaction. This is a small modification and should not impact the properties of curcumin while introducing an easily accessible reactive amino group. This novel compound could be used to couple curcumin to other molecules using the standard methods of peptide synthesis. We studied the aminoethyl-curcumin compound and a tripeptide carrying this aminoethyl-curcumin and the fluorescent dye fluorescein (FITC-curcumin) in vitro on cell culture using confocal laser scanning microscopy and flow cytometry. Then these two substances were tested ex vivo on brain sections prepared from transgenic mice depicting Alzheimer-like ß-amyloid plaques. RESULTS: In the in vitro CLSM microscopy and flow cytometry experiments we found dot-like unspecific uptake and only slight cytotoxicity correlating with this uptake. As these measurements were optimized for the use of fluorescein as dye we found that the curcumin at 488nm fluorescence excitation was not strong enough to use it as a fluorescence marker in these applications. In the ex vivo sections CLSM experiments both the aminoethyl-curcumin and the FITC-curcumin peptide bound specifically to ß- amyloid plaques. CONCLUSION: In conclusion we successfully produced a novel curcumin derivative which could easily be coupled to other imaging or therapeutic molecules as a sensor for amyloid plaques.

A Novel Fluorescence-Labeled Curcumin Conjugate: Synthesis, Evaluation and Imaging on Human Cell Lines.

Curcumin, as the main ingredient of the curcuma spice, has increasingly become the target of scientific research. The turmeric root where the spice is obtained from has been widely used in the traditional medicine. Moreover, scientific studies have found that curcumin has anti-inflammatory, anti-cancer, anti-angiogenic effects as well as antibacterial properties. Recently, curcumin has gathered interest as a potential therapeutic agent in the research on Alzheimer's disease. A consistent problem in the investigative and therapeutic applications of curcumin is its poor solubility in aqueous solutions. In the present study, we synthesized a conjugate of curcumin, the amino acid lysine and the fluorescent dye fluorescein. This conjugate was soluble in cell culture medium and facilitated the examination of curcumin with fluorescence imaging methods. We studied the cell growth impact of unmodified curcumin on seven different human cell lines and then analyzed the uptake and cellular localization of our curcumin conjugate with confocal laser scanning imaging and flow cytometry on the seven cell lines. We found that unbound curcumin inhibited cell growth in vitro and was not taken up into the cells. The curcumin conjugate was internalized into the cell cytoplasm in a dot-like pattern and cellular uptake correlated with the cell membrane damage which was measured using propidium iodide. The CAL-72 osteosarcoma cell exhibited 3-4fold increased conjugate uptake and a strong uniform fluorescein staining in addition to the dot-like pattern observed in all cell lines. In conclusion, we successfully synthesized a novel water-soluble fluorescent curcumin conjugate which showed a strong preference for CAL-72 osteosarcoma cells in vitro.

Fluorescein-labeled Bacitracin and Daptomycin Conjugates: Synthesis, Fluorescence Imaging and Evaluation.

BACKGROUND: Previously, glycopeptides antibiotics such as vancomycin, ramoplanin and an antifungal antibiotic nystatin have been studied for their diagnostic and therapeutic potential. OBJECTIVE: To further explore the diagnostic and chemotherapeutic potential of other antibiotics we have now employed daptomycin, a lipopetide antibiotic and bacitracin, a polypeptide antibiotic in uptake and vitality tests on human cell lines. METHOD: Fluorescent conjugates of bacitracin and daptomycin were synthesized using fluorescein isothiocynate (FITC) for confocal laser scanning microscopy (CLSM) and fluorescence activated cell sorting (FACS). The cellular uptake of the synthesized daptomycin and bacitracin conjugates was studied on seven human cell lines, two healthy and five malignant using CLSM and FACS. To examine the cell membrane damage caused by the conjugates FACS experiments were carried out using propidium iodide. RESULTS: The uptake pattern was different for both antibiotics for all the cell lines. The cytoplasmic uptake of daptomycin conjugate was lower than the bacitracin conjugate, resulting in decreased cell membrane damage. CONCLUSION: No preferential uptake into malignant or healthy cells was found for the two different antibiotic conjugates and the uptake patterns were also different between the two antibiotics. However, the lower cytotoxicity and different uptake mechanism makes daptomycin conjugate a prospective candidate for further study as a diagnostic agent for various intracellular infections.

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.

Fluorescence imaging of human cells with a novel conjugate of the antifungal nystatin.

The antitumor activity of antibacterial and antifungal compounds has been of interest in the past. In several investigations glycopeptide antibiotics like bleomycin and antifungal agents like itraconazole have shown direct positive results whereas antifungal polyenes such as amphotericin B have been shown to potentiate the effects of antitumor agents. After having investigated the fluorescence-marked antibacterial glycopeptides vancomycin and ramoplanin on various malignant and healthy human cells in previous studies, the present work is focused on the antifungal polyene nystatin. We coupled nystatin to the fluorescent dye fluorescein isothiocyanate (FITC). After confirming the correct mass by mass spectrometry the effect of the conjugate on nine different human cell lines (two benign and seven tumor cell lines) was examined. The character of the uptake was determined by confocal laser scanning microscopy (CLSM) and the uptake was quantified by fluorescence activated cell sorting (FACS). The addition of propidium iodide (PI) allowed for detection and quantification of cell membrane disruption caused by the fluorescein-nystatin conjugate. Uptake of the conjugate was found to vary among the nine cell lines investigated. Conjugate uptake was strongest after 6 hours in most cell lines. Only the two prostate carcinoma cell lines PC3 and LNCaP showed further increase in uptake after long-time (24h) incubation. PI staining in general correlated well with the conjugate FITC staining values. The Colo205 colon carcinoma cell line and the U373 and LN18 glioblastoma cell lines exhibited very low conjugate uptake and PI staining. The results indicate that this conjugate shows potential for future imaging studies on certain human cancer cells.

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.

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.

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.

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.

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.

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.

A novel lily-of-the-valley fragrance contrast agent for magnetic resonance and fluorescence imaging of prostate cancer cells.

Detection of prostate carcinoma metastases is currently performed either via indirect tests like the prostate specific antigen (PSA) or prostate cancer gene 3 (PCA3) or by biopsies from masses found with medical imaging methods. Our goal was to use an ectopic odorant receptor to target prostate-derived cells throughout the body for imaging by magnetic resonance and fluorescence imaging. We synthesized a conjugate containing undecylic aldehyde (an antagonist of the human olfactory receptor hOR17-4), gadolinium-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (a common magnetic resonance contrast agent) and fluorescein isothiocyanate (a fluorescent dye). Two different prostate cancer cell lines as well as five other different malignant cell lines and healthy prostate epithelial cells were incubated with this conjugate and evaluated by flow cytometry, confocal laser scanning microscopy and magnetic resonance imaging. The prostate- derived healthy and malignant cells showed stronger fluorescence than the non-prostate cancer cell lines in the flow cytometry and confocal laser scanning microscopy experiments. In the magnetic resonance imaging experiments the T1 relaxation time reduction (higher signal intensity) was also stronger for the prostate-derived cells than for the non-prostate cells. The examined conjugate showed high prostate-cell-specificity. This property makes it of potential value in the diagnosis of prostate cancer lymph node metastases.

Magnetic resonance imaging of human glioma cells by means of an interleukin-6 receptor-targeted contrast agent.

The interleukin 6 receptor (IL-6R) and its ligand interleukin 6 (IL-6) play a crucial role in glioma growth and development accomplished by autocrine growth promotion and induction of angiogenesis via activation of vascular epithelial growth factor A (VEGF-A). Therefore, IL-6R represents a target for both therapy (preventing VEGF-A activation by blocking the receptor) and imaging (higher receptor density on tumor cells). A short heptapeptide that selectively binds to IL-6R and which inhibits the effect of IL-6 was coupled to the magnetic resonance imaging (MRI) contrast agent gadolinium (Gd)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and the fluorescent dye rhodamine. MRI, confocal laser scanning microscopy, and flow cytometry showed that our IL-6-DOTA-rhodamine conjugate was taken up into the cytoplasm of human U373 glioma cells without any cytotoxic effects. Competition experiments indicate that this uptake was receptor-mediated. This conjugate might be used for future MRI studies of brain tumors after systemic or intraoperative local application. The cytoplasm specificity of the conjugate also makes it a potential building block for the design of future cytoplasm-directed imaging and therapeutic conjugates.

Imaging of human glioma cells by means of a Syndecan-4 directed DOTA-conjugate.

The extracellular glycoprotein Tenascin-C (TN-C) is highly upregulated in gliomas. Therefore, many chemotherapies with radiolabeled antibodies against TN-C have been performed. However, TN-Cs binding partner Syndecan-4 did not play any role as a therapeutic or imaging target in gliomas. We constructed an imaging compound containing the magnetic resonance imaging (MRI) contrast agent gadolinium (Gd)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), the fluorescence dye sulforhodamine and a synthetic Syndecan-4-specific 21 amino acid peptide derived from TN-C. Magnetic resonance relaxometry, confocal laser scanning microscopy, and flow cytometry showed that the Syndecan-4-DOTA-Rhodamine conjugate was taken up into the cytoplasm of human U373 glioma cells without any cytotoxic effects. Competition experiments indicate that this uptake was receptor-mediated. This conjugate might be used for future MRI studies of brain tumors after systemic or intraoperative local application.

The lily-of-the-valley fragrance receptor--potential in prostate cancer imaging.

BACKGROUND: The early diagnosis of prostate cancer and its metastases still remains a great challenge. Recently, olfactory receptors have been found in healthy and malignant prostate tissue. If conjugated to a dye, olfactory receptor ligands would represent candidates for markers of not only olfactory cells but also prostate tissue. Such a conjugate would be of great value for the detection of prostate cancer metastases. METHODS: We coupled sulforhodamine, a fluorescence dye, to undecylic aldehyde, the antagonist of the lily-of-the-valley fragrance receptor. By using confocal laser scanning microscopy and flow cytometry we examined the uptake of this conjugate into various different human cell lines. One healthy prostate cell line, two different prostate carcinoma cell lines, and five other carcinoma cell lines were investigated. CD1 nude mice bearing human PC3 prostate carcinoma xenografts were injected with the conjugate. Rhodamine fluorescence of mouse organ frozen sections was evaluated by confocal laser scanning microscopy. RESULTS: When comparing the seven human cell lines, the conjugate was preferentially taken up by the cytoplasm of healthy and malignant prostate cells. The mice showed high conjugate uptake into the xenografts, but much lower uptake into the mouse organs. After 3 hr of circulation, efflux could be observed in the xenograft sections. Xenograft touch prints confirmed in vivo intracellular accumulation. CONCLUSION: This conjugate may be of potential value in the diagnosis of prostate cancer and its metastases.

Cell nucleus directed 2,3,5-triiodobenzoic acid conjugates.

Triiodobenzoic acid (TIBA) represents the core structure of most clinically used contrast agents for computed tomography and other X-ray procedures. To construct an intracellular radiopaque contrast agent, TIBA was coupled to various different positively and negatively charged fluorescein iothiocyanate (FITC)-labelled peptides. TIBA coupled to the SV40 T Antigen nuclear localization sequence (NLS) stained 80% of human glioma cells and caused cell death. This occurred with C- or N-terminal binding of TIBA and with the correct or mutant NLS. No cell death and only small numbers of stained cells (below 3 %) were observed after incubation with NLS conjugates lacking TIBA or after incubation with TIBA-conjugates containing a negatively charged polyglutamic acid stretch. TIBA-conjugates containing the Antennapedia-derived cell-penetrating peptide penetratin were only nuclearly taken up when TIBA and FITC were coupled to lysines outside the 16-amino acid peptide sequence. The study shows that intracellular TIBA may have potential as a chemotherapeutic agent rather than a contrast agent.

The fluorinated and chlorinated nuclear localization sequence of the SV 40 T antigen.

Nuclear uptake of the simian virus (SV) 40 T antigen is triggered by a specific nuclear localization sequence. However, such a nuclear localization sequence is only poorly taken up by the cytoplasm of cells when administered to the culture medium. Our aim was to improve the cytoplasmic uptake of the SV 40 T antigen nuclear localization sequence. Consequently, we synthesized novel fluorescein isothiocyanate-labelled conjugates containing the nuclear localization sequences of the SV 40 T antigen and either trichlorobenzoic or trifluorobenzoic acid. Applied at 260 microM such halogenated NLS conjugates were nuclearly taken up by 75-85% of U373 and LN18 glioma cells and resulted in cell death. Nuclear staining and cell death were also found at lower concentrations (130 and 65 microM) of halogenated nuclear localization sequence conjugates. By contrast only a low cellular staining rate and no cell death could be observed after co-incubation with a trichlorobenzoic acid or trifluorobenzoic acid-lacking nuclear localization sequence conjugate and free, unbound trichlorobenzoic acid or trifluorobenzoic acid at the high concentration (260 microM). Such small non-radioactive fluorinated and chlorinated nuclear localization sequences may be used as important components for future antiglioma drug development.

Value of apoptin's 40-amino-acid C-terminal fragment for the differentiation between human tumor and non-tumor cells.

Apoptin, a protein of the chicken anemia virus (CAV), consists of 121 amino acids (aa) and represents a novel, potentially tumor-specific therapeutic and diagnostic agent. The C-terminal part of Apoptin (aa 81-121) is believed to contain a bipartite nuclear localization signal (NLS) (NLS1: aa 82-88 and NLS2: aa 111-121), which is only active in tumor cells after phosphorylation of threonine(108) by tumor-specific cytoplasmic phosphokinases. Furthermore, a nuclear export signal (NES) (aa 97-105) seems to enable nuclear export of Apoptin only in healthy cells. The specificity for tumor cell nuclei also applies to the truncated C-terminal part of Apoptin (aa 81-121), which therefore represents a highly attractive peptide sequence for peptide synthesis. Here we describe for the first time the synthesis of fluorescein isothiocyanate (FITC)- and Dansyl-labelled conjugates containing this C-terminal part of Apoptin, with either phosphorylated or nonphosphorylated threonine(108). The phosphorylated conjugates were synthesized in an attempt to achieve nuclear accumulation in healthy cells, which lack cytoplasmic tumor-specific phosphokinases. Surprisingly, all the conjugates accumulated rapidly within the cell nuclei of both tumor and non-tumor cells from the bladder, brain and prostate and led to cell death. By coupling Apoptin(81-121) to FITC and DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) at either the C- or N-terminus we could exlude that the coupling site is decisive for tumor cell-specific nuclear localization. The labels FITC, DOTA and Dansyl were not responsible for cell death in healthy cells because cell death was not prevented by using an unlabelled Apoptin(81-121) peptide. Cellular and nuclear uptake of the FITC-labelled Apoptin(81-121) peptide was almost completely abolished after altering the NLS2 (replacement of five arginines with serines).