Targeting the hexosamine biosynthetic pathway and O-linked N-acetylglucosamine cycling for therapeutic and imaging capabilities in diffuse large B-cell lymphoma.

The hexosamine biosynthetic pathway (HBP) requires two key nutrients glucose and glutamine for O-linked N-acetylglucosamine (O-GlcNAc) cycling, a post-translational protein modification that adds GlcNAc to nuclear and cytoplasmic proteins. Increased GlcNAc has been linked to regulatory factors involved in cancer cell growth and survival. However, the biological significance of GlcNAc in diffuse large B-cell lymphoma (DLBCL) is not well defined. This study is the first to show that both the substrate and the endpoint O-GlcNAc transferase (OGT) enzyme of the HBP were highly expressed in DLBCL cell lines and in patient tumors compared with normal B-lymphocytes. Notably, high OGT mRNA levels were associated with poor survival of DLBCL patients. Targeting OGT via small interference RNA in DLBCL cells inhibited activation of GlcNAc, nuclear factor kappa B (NF-κB), and nuclear factor of activated T-cells 1 (NFATc1), as well as cell growth. Depleting both glucose and glutamine in DLBCL cells or treating them with an HBP inhibitor (azaserine) diminished O-GlcNAc protein substrate, inhibited constitutive NF-κB and NFATc1 activation, and induced G0/G1 cell-cycle arrest and apoptosis. Replenishing glucose-and glutamine-deprived DLBCL cells with a synthetic glucose analog (ethylenedicysteine-N-acetylglucosamine [ECG]) reversed these phenotypes. Finally, we showed in both in vitro and in vivo murine models that DLBCL cells easily take up radiolabeled technetium-99m-ECG conjugate. These findings suggest that targeting the HBP has therapeutic relevance for DLBCL and underscores the imaging potential of the glucosamine analog ECG in DLBCL.

99mTc-N4amG: synthesis biodistribution and imaging in breast tumor-bearing rodents.

(99m)Tc-N4-guanine ((99m)Tc-N4amG) was synthesized and evaluated in this study. Cellular uptake and cellular fraction studies were performed to evaluate the cell penetrating ability. Biodistribution and planar imaging were conducted in breast tumor-bearing rats. Up to 17%ID uptake was observed in cellular uptake study with 40% of (99m)Tc-N4amG was accumulated in the nucleus. Biodistribution and scintigraphic imaging studies showed increased tumor/muscle count density ratios as a function of time. Our results demonstrate the feasibility of using (99m)Tc-N4amG in tumor specific imaging.

Development of (99m)Tc-N4-NIM for molecular imaging of tumor hypoxia.

The nitro group of 2-nitroimidazole (NIM) enters the tumor cells and is bioreductively activated and fixed in the hypoxia cells. 1,4,8,11-tetraazacyclotetradecane (N4) has shown to be a stable chelator for (99m)Tc. The present study was aimed to develop (99m)Tc-cyclam-2-nitroimidazole ((99m)Tc-N4-NIM) for tumor hypoxia imaging. N4-NIM precursor was synthesized by reacting N4-oxalate and 1,3-dibromopropane-NIM, yielded 14% (total synthesis). Cell uptake of (99m)Tc-N4-NIM and (99m)Tc-N4 was obtained in 13762 rat mammary tumor cells and mesothelioma cells in 6-well plates. Tissue distribution of (99m)Tc-N4-NIM was evaluated in breast-tumor-bearing rats at 0.5-4 hrs. Tumor oxygen tension was measured using an oxygen probe. Planar imaging was performed in the tumor-bearing rat and rabbit models. Radiochemical purity of (99m)Tc-N4-NIM was >96% by HPLC. Cell uptake of (99m)Tc-N4-NIM was higher than (99m)Tc-N4 in both cell lines. Biodistribution of (99m)Tc-N4-NIM showed increased tumor-to-blood and tumor-to-muscle count density ratios as a function of time. Oxygen tension in tumor tissue was 6-10 mmHg compared to 40-50 mmHg in normal muscle tissue. Planar imaging studies confirmed that the tumors could be visualized clearly with (99m)Tc-N4-NIM in animal models. Efficient synthesis of N4-NIM was achieved. (99m)Tc-N4-NIM is a novel hypoxic probe and may be useful in evaluating cancer therapy.

Molecular imaging of mesothelioma with (99m)Tc-ECG and (68)Ga-ECG.

We have developed ethylenedicysteine-glucosamine (ECG) as an alternative to (18)F-fluoro-2-deoxy-D-glucose ((18)F-FDG) for cancer imaging. ECG localizes in the nuclear components of cells via the hexosamine biosynthetic pathway. This study was to evaluate the feasibility of imaging mesothelioma with (99m)Tc-ECG and (68)Ga-ECG. ECG was synthesized from thiazolidine-4-carboxylic acid and 1,3,4,6-tetra-O-acetyl-2-amino-D-glucopyranose, followed by reduction in sodium and liquid ammonia to yield ECG (52%). ECG was chelated with (99m)Tc/tin (II) and (68)Ga/(69)Ga chloride for in vitro and in vivo studies in mesothelioma. The highest tumor uptake of (99m)Tc-ECG is 0.47 at 30 min post injection, and declined to 0.08 at 240 min post injection. Tumor uptake (%ID/g), tumor/lung, tumor/blood, and tumor/muscle count density ratios for (99m)Tc-ECG (30-240 min) were 0.47 ± 0.06 to 0.08 ± 0.01; 0.71 ± 0.07 to 0.85 ± 0.04; 0.47 ± 0.03 to 0.51 ± 0.01, and 3.49 ± 0.24 to 5.06 ± 0.25; for (68)Ga-ECG (15-60 min) were 0.70 ± 0.06 to 0.92 ± 0.08; 0.64 ± 0.05 to 1.15 ± 0.08; 0.42 ± 0.03 to 0.67 ± 0.07, and 3.84 ± 0.52 to 7.00 ± 1.42; for (18)F-FDG (30-180 min) were 1.86 ± 0.22 to 1.38 ± 0.35; 3.18 ± 0.44 to 2.92 ± 0.34, 4.19 ± 0.44 to 19.41 ± 2.05 and 5.75 ± 2.55 to 3.33 ± 0.65, respectively. Tumor could be clearly visualized with (99m)Tc-ECG and (68)Ga-ECG in mesothelioma-bearing rats. (99m)Tc-ECG and (68)Ga-ECG showed increased uptake in mesothelioma, suggesting they may be useful in diagnosing mesothelioma and also monitoring therapeutic response.

Sulfonylurea receptor as a target for molecular imaging of pancreas beta cells with (99m)Tc-DTPA-glipizide.

OBJECTIVE: This study was aimed to assess pancreas beta cell activity using (99m)Tc-diethyleneaminepentaacetic acid-glipizide (DTPA-GLP), a sulfonylurea receptor agent. The effect of DTPA-GLP on the blood glucose level in rats was also evaluated. METHODS: DTPA dianhydride was conjugated with GLP in the presence of sodium amide, yielding 60%. Biodistribution and planar images were obtained at 30-120 min after injection of (99m)Tc-DTPA-GLP (1 mg/rat, 0.74 and 11.1 MBq per rat, respectively) in normal female Fischer 344 rats. The control group was given (99m)Tc-DTPA. To demonstrate pancreas beta cell uptake of (99m)Tc-DTPA-GLP via a receptor-mediated process, a group of rats was pretreated with streptozotocin (a beta cell toxin, 55 mg/kg, i.v.) and the images were acquired at immediately-65 min on day 5 post-treatment. The effect on the glucose levels after a single administration (ip) of DTPA-GLP was compared to glipizide (GLP) for up to 6 h. RESULTS: The structure of DTPA-GLP was confirmed by NMR, mass spectrometry and HPLC. Radiochemical purity assessed by ITLC was >96%. (99m)Tc-DTPA-GLP showed increased pancreas-to-muscle ratios, whereas (99m)Tc-DTPA showed decreased ratios at various time points. Pancreas could be visualized with (99m)Tc-DTPA-GLP in normal rat, however, (99m)Tc-DTPA has poor uptake suggesting the specificity of (99m)Tc-DTPA-GLP. Pancreas beta cell uptake could be blocked by pre-treatment with streptozotocin. DTPA-GLP showed an equal or better response in lowering the glucose levels compared to the existing GLP drug. CONCLUSIONS: It is feasible to use (99m)Tc-DTPA-GLP to assess pancreas beta cell receptor recognition. (99m)Tc-DTPA-GLP may be helpful in evaluating patients with diabetes, pancreatitis and pancreatic tumors.

Synthesis of (99m)Tc-EC-AMT as an imaging probe for amino acid transporter systems in breast cancer.

OBJECTIVE: This study was to develop a (99m)Tc-labeled alpha-methyl tyrosine (AMT) using L,L-ethylenedicysteine (EC) as a chelator and to evaluate its potential in breast tumor imaging in rodents. METHODS: EC-AMT was synthesized by reacting EC and 3-bromopropyl AMT (N-BOC, ethyl ester) in ethanol/potassium carbonate solution. EC-AMT was labeled with (99m)Tc in the presence of tin (II) chloride. Rhenium-EC-AMT (Re-EC-AMT) was synthesized as a reference standard for (99m)Tc-EC-AMT. To assess the cellular uptake kinetics of (99m)Tc-EC-AMT, 13 762 rat breast cancer cells were incubated with (99m)Tc-EC-AMT for 0-2 h. To investigate the transport mechanism, the same cell line was used to conduct the competitive inhibition study using L-tyrosine. Tissue distribution of (99m)Tc-EC-AMT was determined in normal rats at 0.5-4 h. Planar imaging of breast tumor-bearing rats was performed at 30 and 90 min. The data were compared with those of (18)F-2-fluoro-2-deoxy-glucose. Blocking uptake study using unlabeled AMT was conducted to investigate the transport mechanism of (99m)Tc-EC-AMT in vivo. RESULTS: Structures of EC-AMT and Re-EC-AMT were confirmed by nuclear magnetic resonance, high performance liquid chromatography and mass spectra. In-vitro cellular uptake of (99m)Tc-EC-AMT in 13,762 cells was increased as compared with that of (99m)Tc-EC and could be inhibited by L-tyrosine. Biodistribution in normal rats showed high in-vivo stability of (99m)Tc-EC-AMT. Planar scintigraphy at 30 and 90 min showed that (99m)Tc-EC-AMT could clearly visualize tumors. (99m)Tc-EC-AMT uptake could be significantly blocked by unlabeled AMT in vivo. CONCLUSION: The results indicate that (99m)Tc-EC-AMT, a new amino acid transporter-based radiotracer, is suitable for breast tumor imaging.

Imaging and dosimetry of 99mTc EC annexin V: preliminary clinical study targeting apoptosis in breast tumors.

BACKGROUND: Early detection of cellular events is important to predict the outcome of the patients. This study was aimed to use (99m)Tc EC-annexin V to image tumor cells undergoing apoptosis. METHODS: In 10 patients with breast cancer, scintigraphic images and dosimetric estimates were obtained after administering (99m)Tc EC-annexin V. RESULTS: Nine of the 10 cases showed detectable (99m)Tc EC-annexin V uptake in tumor. Higher values of T/N ratios are associated with patient after treatment. CONCLUSIONS: Apoptosis can be quantified using (99m)Tc EC-annexin V.

Radiolabeled L-lysine for tumor imaging.

RATIONALE AND OBJECTIVES: The aims of this study were to label the versatile amino acid l-lysine with (99m)Tc using 2,3-dimercapto-succinic acid (DMSA) as a chelator, and to assess its tumor imaging feasibility under in vivo and in vitro conditions, and finally to determine the subcellular biodistribution of this radiopharmaceutical. MATERIALS AND METHODS: DMSA-l-lysine was chemically synthesized and labeled with sodium pertechnetate. Nuclear magnetic resonance (NMR) and mass spectral analysis of DMSA-l-lysine were conducted. Radiochemical purity was determined by thin-layer chromatography (TLC) and paper chromatography. Cellular uptake, competition and subcellular localization studies were performed in rat breast cancer cells (13762). In vivo studies of planar imaging and biodistribution studies were performed on female Fischer 344 rats. Medical Internal Radiation Dose (MIRD) dosimetry estimates were calculated. RESULTS: Radiochemical purity (determined by radio-TLC and high-performance liquid chromatography) of these compounds was >95%. (99m)Tc-DMSA-l-lysine showed good uptake in in vitro cell culture assays and uptake was reduced in competition studies. (99m)Tc-DMSA-l-lysine accumulates in the nucleus as much as in the cytoplasm and it was also shown that accumulation of the (99m)Tc-DMSA-l-lysine in the nucleus increases as a function of a time. There was an increase in tumor-to-blood and tumor-to-muscle count density ratios. Tumor/background ratios were 5.75 at 1 hour and 6.87 at 2 hours. In vivo tissue distribution studies revealed that radiation dosimetry of blood-forming organs were within radiation dose limits. CONCLUSION: DMSA-l-lysine kits can be labeled with (99m)Tc easily and efficiently, with high radiochemical purity and cost-effectiveness. In vitro cellular uptake and scintigraphic imaging studies demonstrated the pharmacokinetic distribution and feasibility of using (99m)Tc-DMSA-l-lysine for tumor imaging.

PET and planar imaging of tumor hypoxia with labeled metronidazole.

RATIONALE AND OBJECTIVES: This study was aimed to develop 99mTc- and 68Ga-labeled metronidazole (MN) using ethylenedicysteine (EC) as a chelator and evaluate their potential use to assess tumor hypoxia. MATERIALS AND METHODS: EC-MN was labeled with 99mTc in the presence of tin (II) chloride. Labeling EC-MN with 68Ga was achieved by adding 68GaCl3 (2 mCi with 3.4 microg cold GaCl3). In vitro cellular uptakes of 99mTc- and 68Ga-EC-MN were obtained in various types of tumor cells at 0.5-4 hours. Tissue distribution and PET imaging of 99mTc and 68Ga-EC-MN were evaluated in breast tumor-bearing rats at 0.5-4 hours. Tumor oxygen tension was measured using an oxygen probe. RESULTS: There were similar cellular uptakes (2-10%) between 99mTc- and 68Ga-EC-MN at 0.5-4 hours. In vivo biodistribution of 99mTc- and 68Ga-EC-MN in breast tumor-bearing rats showed increased tumor-to-blood and tumor-to-muscle count density ratios as a function of time. Positron emission tomography images confirmed that the tumors could be visualized clearly with 68Ga-EC-MN. Oxygen tension in tumor tissue was determined to be 6-10 mm Hg compared with 40-50 mm Hg in normal muscle tissue. CONCLUSIONS: The results indicated that it is feasible to use 99mTc- and 68Ga-EC-MN for assessment of tumor hypoxia. These agents may be useful in selecting and evaluating cancer therapy.

Regional radiochemotherapy using in situ hydrogel.

PURPOSE: To evaluate the feasibility of regional radiochemotherapy of mammary tumors using in situ hydrogel loaded with cisplatin (CDDP) and rhenium-188 ((188)Re). METHODS: Sodium alginate (SA) and calcium chloride were used to create a hydrogel for delivery of CDDP and (188)Re. In vitro studies were performed to evaluate cytotoxic effects of (188)Re-hydrogel and sustained-release ability of the CDDP-hydrogel. Tumor-bearing rats were injected with (188)Re-hydrogel (0.5-1 mCi/rat), (188)Re-perrhenate (0.5-1 mCi/rat, intratumoral, I.T.), CDDP-hydrogel (3 mg/kg), and (188)Re-hydrogel loaded with CDDP (3 mg/kg body weight, 0.5-1 mCi/rat), respectively, and groups receiving (188)Re were imaged at 24 and 48 h postinjection. Tumor volume, body weight, imaging, and kidney function were assessed as required for each group. RESULTS: Successful formation of the hydrogel was demonstrated by cytotoxic effects of (188)Re-hydrogel and slow release of CDDP-hydrogel in vitro. Tumor volume measurements showed significant delay in tumor growth in treated vs. control groups with minimal variation in normal kidney function for the CDDP-hydrogel group. Scintigraphic images indicated localization of (188)Re-hydrogel in the tumor site up to 48 h postinjection. CONCLUSIONS: Our data demonstrate the feasibility of using hydrogel for delivery of chemotherapeutics and radiation locally. This technique may have applications involving other contrast modalities as well as treatment in cases where tumors are inoperable.

Assessment of cyclooxygense-2 expression with 99mTc-labeled celebrex.

Cyclooxygenase-2 (COX-2) plays an important role in angiogenesis and cancer progression. Since many tumor cells exhibit COX-2 expression, functional imaging of COX-2 expression using celebrex (CBX, a COX-2 inhibitor) may provide not only a non-invasive, reproducible, quantifiable alternative to biopsies, but it also greatly complements pharmacokinetic studies by correlating clinical responses with biological effects. Moreover, molecular endpoints of anti-COX-2 therapy could also be assessed effectively. This study aimed at measuring uptake of Tc-EC-CBX in COX-2 expression in tumor-bearing animal models. In vitro Western blot analysis and cellular uptake assays were used to examine the feasibility of using Tc-EC-CBX to measure COX-2 activity. Tissue distribution studies of Tc-EC-CBX were evaluated in tumor-bearing rodents at 0.5-4 h. Dosimetric absorption was then estimated. Planar scintigraphy was performed in mice, rats and rabbits bearing tumors. In vitro cellular uptake indicated that cells with higher COX-2 expression (A549 and 13762) had higher uptake of Tc-EC-CBX than lower COX-2 expression (H226). In vivo biodistribution of Tc-EC-CBX in tumor-bearing rodents showed increased tumor:tissue ratios as a function of time. In vitro and biodistribution studies demonstrated the possibility of using Tc-EC-CBX to assess COX-2 expression. Planar images confirmed that the tumors could be visualized with Tc-EC-CBX from 0.5 to 4 h in tumor-bearing animal models. We conclude that Tc-EC-CBX may be useful to assess tumor COX-2 expression. This may be useful in the future for selecting patients for treatment with anti-COX-2 agents.

Alternative splicing disrupts a nuclear localization signal in spleen tyrosine kinase that is required for invasion suppression in breast cancer.

Spleen tyrosine kinase (Syk) is a candidate tumor (metastasis) suppressor that is highly expressed in mammary epithelial cells. Loss of Syk expression through promoter hypermethylation is associated with increased invasiveness in a subset of breast cancer. Here, we show that in addition to full-length Syk [Syk(L)], an alternatively spliced variant, Syk(S), is frequently expressed in breast cancer cells. Syk(S) is identical to Syk(L), except that it lacks 23 amino acid residues (deletion) within the interdomain B (IDB) of Syk. We also show that the aberrant expression of Syk(S) occurs frequently in primary breast tumors but never in matched normal mammary tissues, suggesting a contribution of Syk(S) to mammary tumor progression. Expression of Syk(L) suppressed breast cancer cell invasiveness. In contrast, Syk(S) expression did not affect the cell invasion potential. This differential phenotypic response is accompanied by their different subcellular localization. Immunocytochemical studies and nuclear and cytoplasmic fractionation experiments indicated that Syk(L) could enter the nucleus, whereas Syk(S) was located exclusively in the cytoplasm. Five basic residues in deletion were found to be critical in determining Syk(L) nuclear transport and invasion suppression activity; mutations completely excluded Syk(L) from the nucleus and blocked Syk(L)-inducible invasion suppression. Moreover, IDB acted as an autonomous nuclear localization signal to facilitate nuclear transport of a heterologous protein. Thus, the IDB of Syk(L) contains a nuclear localization signal that is responsible for Syk(L) nuclear translocation. The correlation of the nuclear localization and invasion suppression function of Syk(L) indicated that nuclear Syk possesses biological activities associated with tumor suppression in mammary epithelial cells.

TRATAMIENTO PARA DESDENTADOS PARCIALES CLASE II DE KENNEDY DEL MAXILAR INFERIOR CON PROTESIS PARCIAL REMOVIBLE SOBREPUESTA

Se logró rescatar aquellos dientes con graves lesiones cariosas y compromiso pulpar para recibir las coronas primarias. Se logró dar utilidad con pilares, aquellos dientes con tratamiento de conductos, mediante la reconstrucción con resina fotopolimerizable. Se consiguio dar utilidad como pilares de anclaje, a quellos dientes en estado radicular contratamiento y sin tratamiento de conductos, mediante la confección de pernos muñones primarios. Se permite una distribución uniformede las fuerzas verticales, a través de la estructura rígida hacia los pilares dentales y el reborde residual, mejorando de esta manera el soporte dentomucoso. Se consiguio incrementar la retención en la clase II de kennedy gracias a las coronas telescópicas y ganchos accesorios. Se logro una mayor estabilidad de la prótesis evitando los movimientos en el extremo libre, gracias a los directos (coronas telescópicas) e indirectos a manera de apuntamiento, cobyubados por la barra lingual de naturaleza semirígida y de la estructura en general. Se mejoro la estética en el sector anterior eliminando por completo los ganchos en zona crítica. Lo que lleva a una aceptación por parte del paciente