carbocyanines and Prostatic-Neoplasms

This review covers the application of heptamethine cyanine dye (HMCD) mediated drug delivery. A relatively small number of HMCDs possess tumor targeting abilities, and this has spurred interest from research groups to explore them as drug delivery systems. Their tumor selectivity is primarily attributed to their uptake by certain isoforms of organic anion transporting polypeptides (OATPs) which are overexpressed in cancer tissues, although there are other possible mechanisms for the observed selectivity still under investigation. This specificity is confirmed using various cancer cell lines and is accompanied by moderate cytotoxicity. Their retention in tumor tissue is facilitated by the formation of albumin adducts as revealed by published mechanistic studies. HMCDs are also organelle selective dyes with specificity toward mitochondria and lysosomes, and with absorption and emission in the near-infrared region. This makes them valuable tools for biomedical imaging, especially in the field of fluorescence-guided tumor surgery. Furthermore, conjugating antitumor agents to HMCDs is providing novel drugs that await clinical testing. HMCD development as theranostic agents with dual tumor targeting and treatment capability signals a new approach to overcome drug resistance (mediated through evasion of efflux pumps) and systemic toxicity, the two parameters which have long plagued drug discovery.

Topics: Antineoplastic Agents; Brain Neoplasms; Breast Neoplasms; Burkitt Lymphoma; Carbocyanines; Coloring Agents; Drug Delivery Systems; Drug Discovery; Drug Resistance, Neoplasm; Female; Humans; Kidney Neoplasms; Male; Precision Medicine; Prostatic Neoplasms

Prostate cancer (PCa) is considered to be the most prevalent malignancy in males worldwide. Abiraterone is a 17α-hydroxylase/C17, 20-lyase (CYP17) inhibitor that has been approved for use in patients with prostate cancer. However, several negative aspects, such as drug resistance, toxicity, and lack of real-time monitoring of treatment responses, could appear with long-term use. Therefore, the development of anticancer agents with specific targeting to avoid side effects is imperative. Here, we used MHI-148, a type of heptamethine cyanine (HC) near-infrared fluorescence dye (NIRF), as a prototype structure to synthesize two theranostic agents, Abi-DZ-1 and Abi-783. The new compound Abi-DZ-1 retained the excellent photophysical characteristics and NIRF imaging property of MHI-148, and it could preferentially accumulate in prostate cancer cells but not in normal prostate epithelial cells via the HIF1α/organic anion-transporting polypeptides axis. NIRF imaging using Abi-DZ-1 selectively identified tumors in mice bearing PCa xenografts. Moreover, Abi-DZ-1 treatment significantly retarded the tumor growth in both a cell-derived xenograft model and a patient-derived tumor xenograft model. This finding demonstrated that Abi-DZ-1 may hold promise as a potential multifunctional theranostic agent for future tumor-targeted imaging and precision therapy. Constructing theranostic agents using the NIRF dye platform holds great promise in accurate therapy and intraoperative navigation.

Topics: Animals; Carbocyanines; Cell Line, Tumor; Fluorescent Dyes; Humans; Male; Mice; Mitochondria; Organic Anion Transporters; Prostatic Neoplasms

Recent studies have shown that monoamine oxidase A (MAOA) is significantly expressed in malignant prostate cancer (PCa) and plays an important role in tumorigenesis indicating its potential to serve as a target for PCa treatment. Here, we choose the small molecule isoniazid as the MAOA inhibition functionality and incorporated it in the tumor-targeting moiety of heptamethine carbocyanine dyes

Topics: Antineoplastic Agents; Carbocyanines; Cell Line, Tumor; Drug Delivery Systems; Humans; Hydrogen-Ion Concentration; Isoniazid; Male; Monoamine Oxidase Inhibitors; Prostatic Neoplasms

Direct and absolute analysis of microRNAs (miRNAs) in complex media (e.g., human serum) is still a big challenge due to the issues with off-analyte absorption, low sensitivity and specificity. In this work, we have fabricated the erythrocyte membrane-biointerfaced spherical nucleic acids (EMSNAs) for miRNA assay, which not only enables tailor-engineered signal amplification but also exhibits anti-interference property. As a consequence, it is possible to achieve a single-step quantification of miRNAs in complex media without the process of enzymatic amplification, which can vastly simplify the detection procedure. Experimental results reveal that the assay permits ultrasensitive quantification of miR-141, with a limit of detection down to 33.9 aM, and show a high selectivity for discriminating miR-200 family members. More importantly, the assay enables robust miRNA analysis in human serum and can accurately differentiate lung cancer patients and prostate cancer patients from healthy donors. Its performance may satisfy the requirements for direct, rapid, sensitive and specific early diagnosis of cancer, signifying its great potential in clinical diagnostics.

Topics: Animals; Carbocyanines; DNA Probes; Erythrocyte Membrane; Fluorescent Dyes; Gold; Humans; Limit of Detection; Lung Neoplasms; Male; Mice, Inbred BALB C; MicroRNAs; Nanostructures; Nucleic Acid Hybridization; Prostatic Neoplasms; Spectrometry, Fluorescence

Antibody-based dual-modality (PET/fluorescence) imaging enables both presurgery antigen-specific immuno-PET for noninvasive whole-body evaluation and intraoperative fluorescence for visualization of superficial tissue layers for image-guided surgery.

Topics: Animals; Antibodies; Antigens, Neoplasm; Carbocyanines; Cyclooctanes; Cysteine; Fluorescence; Fluorescent Dyes; Fluorine Radioisotopes; GPI-Linked Proteins; Humans; Immunoglobulin Fragments; Male; Mice; Microscopy, Fluorescence; Neoplasm Proteins; Neoplasm Transplantation; Optical Imaging; Positron-Emission Tomography; Prostatic Neoplasms; Radiopharmaceuticals; Tissue Distribution

Monoamine oxidase A (MAOA) is an important mitochondria-bound enzyme that catalyzes the oxidative deamination of monoamine neurotransmitters. Accumulating evidence suggests a significant association of increased MAOA expression and advanced high-grade prostate cancer (PCa) progression and metastasis. Herein, a series of novel conjugates combining the MAOA inhibitor isoniazid (INH) and tumor-targeting near-infrared (NIR) heptamethine cyanine dyes were designed and synthesized. The synthesized compounds

Topics: Carbocyanines; Fluorescent Dyes; Humans; Isoniazid; Male; Molecular Docking Simulation; Monoamine Oxidase; Monoamine Oxidase Inhibitors; Neoplasm Proteins; PC-3 Cells; Prostatic Neoplasms

Clinical trials involving genome-edited cells are growing in popularity, where CAR-T immunotherapy and CRISPR/Cas9 editing are more recognized strategies. Genetic reporters are needed to localize the molecular events inside these cells in patients. Specifically, a nonimmunogenic genetic reporter is urgently needed as current reporters are immunogenic due to derivation from nonhuman sources. Prostate-specific membrane antigen (PSMA) is potentially nonimmunogenic due to its natural, low-level expression in select tissues (self-MHC display). PSMA overexpression on human prostate adenocarcinoma is also visible with excellent contrast. We exploit these properties in a transduced, two-component,

Topics: Animals; Antigens, Surface; Carbocyanines; Cell Line, Tumor; Cell Tracking; Fluorescent Dyes; Genes, Reporter; Glutamate Carboxypeptidase II; Humans; Male; Mice; Models, Molecular; Optical Imaging; Positron-Emission Tomography; Prostatic Neoplasms

Prostate-specific membrane antigen (PSMA) is an important biological target for therapy and diagnosis of prostate cancer. In this study, novel multivalent PSMA inhibitors with glutamate-urea-lysine structures were designed to improve inhibition characteristics. Precursors of the novel inhibitors were prepared from glutamic acid with di-tert-butyl ester. A near-infrared molecular dye, sulfo-Cy5.5, was introduced into the precursors to generate the final PSMA fluorescent inhibitors, compounds 12-14, to visualize prostate cancer. Biological behaviors of the inhibitors were evaluated using in vitro inhibition assays, in vivo fluorescent imaging, and ex vivo biodistribution assays. K

Topics: Animals; Antineoplastic Agents; Carbocyanines; Female; Fluorescent Dyes; Glutamate Carboxypeptidase II; Male; Membrane Glycoproteins; Mice, Inbred BALB C; Prostatic Neoplasms; Tissue Distribution

The presence of positive surgical margins confers an increased risk of biochemical relapse and need for salvage therapy in men undergoing radical prostatectomy. Image-guided surgery using near-infrared (NIR) fluorescent contrast agents is a potential method to detect remaining cancerous tissue. The objective of this study was to evaluate three hyaluronic acid (HA) nanoparticle (NP) formulations loaded with NIR fluorophore for their ability to contrast-enhance prostate cancer. HA was modified by conjugation with the hydrophobic ligand, aminopropyl-1-pyrenebutanamide to drive nanoparticle self-assembly. Indocyanine green (ICG) was physicochemically entrapped in the HA-NP, termed NanoICG. Alternatively, Cy7.5 was directly conjugated to amphiphilic HA, termed NanoCy7.5. NanoCy7.5 was synthesized with two HA molecular weights to determine the HA size contribution to delivery to PC3 prostate tumor xenografts. Contrast-enhancement of the tumors and relative biodistribution were assessed by a series of fluorescence imaging, image-guided surgery with spectroscopy, and microscopic techniques. Intravenously administered NanoICG improved tumor signal-to-noise ratio (SNR) at 24 h over ICG by 2.9-fold. NanoCy7.5 with 10 kDa and 100 kDa HA improved tumor SNR by 6.6- and 3.1-fold over Cy7.5 alone, respectively. The PC3 xenograft was clearly identified with the image-guided system providing increased contrast enhancement compared to surrounding tissue for NanoICG and NanoCy7.5 with 10 kDa HA. NIR fluorescence microscopy showed that Cy7.5 in NPs with 10 kDa HA were distributed throughout the tumor, while NanoCy7.5 with 100 kDa HA or NanoICG delivered dye mainly to the edge of the tumor. CD31 staining suggested that PC3 tumors are poorly vascularized. These studies demonstrate the efficacy of a panel of HA-derived NPs in identifying prostate tumors in vivo, and suggest that by tuning the structural properties of these NPs, optimized delivery can be achieved to poorly vascularized tumors.. We have demonstrated the potential of a panel of near-infrared fluorescent (NIRF) nanoparticles (NPs) for image-guided surgery in a prostate cancer xenograft model. Image-guided surgery and imaging of organs ex vivo showed greater tumor signal and contrast when mice were administered NIRF dyes that were covalently conjugated to (NanoCy7.5

Topics: Animals; Carbocyanines; Cell Line, Tumor; Contrast Media; Heterografts; Humans; Hyaluronic Acid; Infrared Rays; Male; Mice; Mice, Nude; Microscopy, Fluorescence; Nanoparticles; Neoplasm Transplantation; Prostatic Neoplasms

Sialylated glycans are found at elevated levels in many types of cancer and have been implicated in disease progression. However, the specific glycoproteins that contribute to the cancer cell-surface sialylation are not well characterized, specifically in bona fide human disease tissue. Metabolic and bioorthogonal labeling methods have previously enabled the enrichment and identification of sialoglycoproteins from cultured cells and model organisms. Herein, we report the first application of this glycoproteomic platform to human tissues cultured ex vivo. Both normal and cancerous prostate tissues were sliced and cultured in the presence of the azide-functionalized sialic acid biosynthetic precursor Ac

Topics: Azides; Biotinylation; Carbocyanines; Hexosamines; Humans; In Vitro Techniques; Male; Mass Spectrometry; Microscopy, Fluorescence; Principal Component Analysis; Prostatic Neoplasms; Proteomics; Sialoglycoproteins; Voltage-Dependent Anion Channel 1

The increasing prevalence of ultra-high-field magnetic resonance imaging (UHFMRI) in biomedical research and clinical settings will improve the resolution and diagnostic accuracy of MRI scans. However, better contrast agents are needed to achieve a satisfactory signal-to-noise ratio. Here, we report the synthesis of a bimodal contrast agent prepared by loading the internal cavity of tobacco mosaic virus (TMV) nanoparticles with a dysprosium (Dy

Topics: Animals; Carbocyanines; Dysprosium; Humans; Integrin alpha2beta1; Magnetic Resonance Imaging; Male; Mice, Nude; Nanoparticles; Oligopeptides; Optical Imaging; PC-3 Cells; Prostatic Neoplasms; Tobacco Mosaic Virus

In this study, the lipid targeted nanobubble carrying the A10-3.2 aptamer against prostate specific membrane antigen was fabricated, and its effect in the ultrasound imaging of prostate cancer was investigated. Materials including 2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol, carboxyl-modified 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, and polyethyleneglycol-2000 were for mechanical oscillation, and nanobubbles were obtained through the centrifugal flotation method. After mice were injected with nanobubbles, abdominal color Doppler blood flow imaging significantly improved. Through left ventricular perfusion with normal saline to empty the circulating nanobubbles, nanobubbles still existed in tumor tissue sections, which demonstrated that nanobubbles could enter tissue spaces via the permeability and retention effect. Fluorinated A10-3.2 aptamers obtained by chemical synthesis had good specificity for PSMA-positive cells, and were linked with carboxyl-modified 1,2-distearoyl-sn-glycero-3-phosphoethanolamine lipid molecules from the outer shell of nanobubbles via amide reaction to construct targeted nanobubbles. Gel electrophoresis and immunofluorescence confirmed that targeted nanobubbles were fabricated successfully. Next, targeted nanobubbles could bind with PSMA-positive cells (C4-2 cells), while not with PSMA-negative cells (PC-3 cells), using in vitro binding experiments and flow cytometry at the cellular level. Finally, C4-2 and PC-3 xenografts in mice were used to observe changes in parameters of targeted and non-targeted nanobubbles in the contrast-enhanced ultrasound mode, and the distribution of Cy5.5-labeled targeted nanobubbles in fluorescent imaging of live small animals. Comparison of ultrasound indicators between targeted and non-targeted nanobubbles in C4-2 xenografts showed that they had similar peak times (P>0.05), while the peak intensity, half time of peak intensity, and area under the curve of ½ peak intensity were significantly different (P<0.05). In PC-3 xenografts, there were no differences in these four indicators. Fluorescent imaging indicated that targeted nanobubbles had an aggregation ability in C4-2 xenograft tumors. In conclusion, targeted nanobubbles carrying the anti-PSMA A10-3.2 aptamer have a targeted imaging effect in prostate cancer.

Topics: Animals; Antigens, Surface; Aptamers, Nucleotide; Carbocyanines; Cell Line, Tumor; Contrast Media; Diagnostic Uses of Chemicals; Glutamate Carboxypeptidase II; Humans; Lipids; Male; Mice, Nude; Nanostructures; Particle Size; Polyethylene Glycols; Prostatic Neoplasms; Ultrasonography; Ultrasonography, Doppler, Color; Xenograft Model Antitumor Assays

Near‑infrared fluorescence (NIRF) imaging is an attractive novel modality for the detection of cancer. A previous study defined two organic polymethine cyanine dyes as ideal NIRF probes, IR‑783 and its derivative MHI‑148, which have excellent optical characteristics, superior biocompatibility and cancer targeting abilities. To investigate the feasibility of NIRF dye‑mediated prostate cancer imaging, dye uptake and subcellular co‑localization were investigated in PC‑3, DU‑145 and LNCaP human prostate cancer cells and RWPE‑1 normal prostate epithelial cells. Different organic anion transporting peptide (OATP) inhibitors were utilized to explore the potential role of the OATP subtype, including the nonspecific OATP inhibitor bromosulfophthalein, the OATP1 inhibitor 17β‑estradiol, the selective OATP1B1 inhibitor rifampicin and the selective OATP1B3 inhibitor cholecystokinin octapeptide. NIRF dyes were also used for the simulated detection of circulating tumor cells and the rapid detection of prostate cancer in human prostate cancer tissues and prostate cancer xenografts in mouse models. The results revealed that the cancer‑specific uptake of these organic dyes in prostate cancer cells occurred primarily via OATP1B3. A strong NIRF signal was detected in prostate cancer tissues, but not in normal tissues that were stained with IR‑783. Prostate cancer cells were recognized with particular NIR fluorescence in isolated mononuclear cell mixtures. The results of the present study demonstrated that NIRF dye‑mediated imaging is a feasible and practicable method for prostate cancer detection, although further investigative studies are required before clinical translation.

Topics: Animals; Carbocyanines; Cell Line; Estradiol; Flow Cytometry; Fluorescent Dyes; Humans; Liver-Specific Organic Anion Transporter 1; Male; Mice; Mice, Nude; Microscopy, Confocal; Neoplastic Cells, Circulating; Organic Anion Transporters; Organic Anion Transporters, Sodium-Independent; Organic Cation Transport Proteins; Prostate; Prostatic Neoplasms; Rifampin; Sincalide; Solute Carrier Organic Anion Transporter Family Member 1B3; Spectroscopy, Near-Infrared; Sulfobromophthalein; Transplantation, Heterologous

In order to enhance visualization of soft tissues, a dual-imaging diagnostic polymersome system featured with highly hydrated multilamellar wall structure capable of simultaneously embedding a hydrophobic near-infrared fluorophore, Cy5.5, and a paramagnetic probe, gadolinium (Gd(III)) cations was developed. The polymersomes were obtained from the self-assembly of lipid-containing copolymer, poly(acrylic acid-co-distearin acrylate), in aqueous solution. The Cy5.5 and Gd(III) species were loaded into polymersomes via hydrophobic association (loading efficiency of Cy5.5 ca 74%) and electrostatic complexation (Gd(III) 83%), respectively. The Cy5.5/Gd(III)-loaded polymersomes (CGLPs) have shown excellent payload confinement, reduced dilution effect on assembly dissociation and decreased protein/salt-induced colloidal aggregation. Owing to the highly hydrated structure of vesicular membrane, the superior contrast enhancement of CGLPs in magnetic resonance (MR) imaging was obtained as a result of prolonged rotational correlation time of Gd(III) cations and fast water exchange from Gd(III) to bulk solution. The CGLPs exhibit a 15-fold higher longitudinal relaxivity value (ca 60 mM(-1) s(-1)) than that (4 mM(-1) s(-1)) of the commercial contrast agent, Magnevist, in phosphate buffered saline. The in vivo characterization demonstrates that CGLPs exhibit a signal-to-noise ratio in T1-weighted MR image contrast similar to that of Magnevist, yet with a Gd dose 5-fold lower. An excellent contrast in NIR imaging at tumor site was attained following the intravenous injection of GGLPs into Tramp-C1 tumor-bearing mice (C57BL/6). Along with their non-toxicity at the dose used, these results demonstrate the great potential of the CGLPs as an advanced diagnostic nanodevice.

Topics: Acrylic Resins; Animals; Biological Transport; Carbocyanines; Cell Survival; Contrast Media; Diglycerides; Drug Delivery Systems; Fluorescent Dyes; Gadolinium; HeLa Cells; Humans; Hydrophobic and Hydrophilic Interactions; Magnetic Resonance Imaging; Male; Mice; Mice, Inbred C57BL; Molecular Imaging; Neoplasm Transplantation; Polymerization; Prostatic Neoplasms

Extradomain-B fibronectin (EDB-FN), one of the oncofetal fibronectin (onfFN) isoforms, is a high-molecular-weight glycoprotein that mediates cell adhesion and migration. The expression of EDB-FN is associated with a number of cancer-related biological processes such as tumorigenesis, angiogenesis, and epithelial-to-mesenchymal transition (EMT). Here, we report the development of a small peptide specific to EDB-FN for targeting prostate cancer. A cyclic nonapeptide, CTVRTSADC (ZD2), was identified using peptide phage display. A ZD2-Cy5 conjugate was synthesized to accomplish molecular imaging of prostate cancer in vitro and in vivo. ZD2-Cy5 demonstrated effective binding to up-regulated EDB-FN secreted by TGF-β-induced PC3 cancer cells following EMT. Following intravenous injections, the targeted fluorescent probe specifically bound to and delineated PC3-GFP prostate tumors in nude mice bearing the tumor xenografts. ZD2-Cy5 also showed stronger binding to human prostate tumor specimens with a higher Gleason score (GS9) compared to those with a lower score (GS 7), with no binding in benign prostatic hyperplasia (BPH). Thus, the ZD2 peptide is a promising strategy for molecular imaging and targeted therapy of prostate cancer.

Topics: Amino Acid Sequence; Animals; Carbocyanines; Cell Line, Tumor; Cell Transformation, Neoplastic; Coloring Agents; Fibronectins; Humans; Male; Mice; Mice, Nude; Molecular Targeted Therapy; Oligopeptides; Optical Imaging; Prostatic Neoplasms; Substrate Specificity; Transforming Growth Factor beta

Brain tumors and brain metastases are among the deadliest malignancies of all human cancers, largely due to the cellular blood-brain and blood-tumor barriers that limit the delivery of imaging and therapeutic agents from the systemic circulation to tumors. Thus, improved strategies for brain tumor visualization and targeted treatment are critically needed. Here we identified and synthesized a group of near-infrared fluorescence (NIRF) heptamethine carbocyanine dyes and derivative NIRF dye-drug conjugates for effective imaging and therapeutic targeting of brain tumors of either primary or metastatic origin in mice, which is mechanistically mediated by tumor hypoxia and organic anion-transporting polypeptide genes. We also demonstrate that these dyes, when conjugated to chemotherapeutic agents such as gemcitabine, significantly restricted the growth of both intracranial glioma xenografts and prostate tumor brain metastases and prolonged survival in mice. These results show promise in the application of NIRF dyes as novel theranostic agents for the detection and treatment of brain tumors.

Topics: Animals; Blood-Brain Barrier; Brain Neoplasms; Carbocyanines; Cell Line, Tumor; Deoxycytidine; Diagnostic Imaging; Drug Delivery Systems; Fluorescent Dyes; Gemcitabine; HEK293 Cells; Humans; Hypoxia; Male; Mice, Nude; Mice, SCID; Neoplasm Metastasis; Organic Anion Transporters; Prostatic Neoplasms; Spectroscopy, Near-Infrared; Xenograft Model Antitumor Assays

The quantitative analysis of foci plays an important role in many cell biological methods such as counting of colonies or cells, organelles or vesicles, or the number of protein complexes. In radiation biology and molecular radiation oncology, DNA damage and DNA repair kinetics upon ionizing radiation (IR) are evaluated by counting protein clusters or accumulations of phosphorylated proteins recruited to DNA damage sites. Consistency in counting and interpretation of foci remains challenging. Many current software solutions describe instructions for time-consuming and error-prone manual analysis, provide incomplete algorithms for analysis or are expensive. Therefore, we aimed to develop a tool for costless, automated, quantitative and qualitative analysis of foci.. For this purpose we integrated a user-friendly interface into ImageJ and selected parameters to allow automated selection of regions of interest (ROIs) depending on their size and circularity. We added different export options and a batch analysis. The use of the Focinator was tested by analyzing γ-H2.AX foci in murine prostate adenocarcinoma cells (TRAMP-C1) at different time points after IR with 0.5 to 3 Gray (Gy). Additionally, measurements were performed by users with different backgrounds and experience.. The Focinator turned out to be an easily adjustable tool for automation of foci counting. It significantly reduced the analysis time of radiation-induced DNA-damage foci. Furthermore, different user groups were able to achieve a similar counting velocity. Importantly, there was no difference in nuclei detection between the Focinator and ImageJ alone.. The Focinator is a costless, user-friendly tool for fast high-throughput evaluation of DNA repair foci. The macro allows improved foci evaluation regarding accuracy, reproducibility and analysis speed compared to manual analysis. As innovative option, the macro offers a combination of multichannel evaluation including colocalization analysis and the possibility to run all analyses in a batch mode.

Topics: Adenocarcinoma; Algorithms; Animals; Automation; Carbocyanines; Cell Count; Data Display; DNA Damage; DNA, Neoplasm; Fluorescent Antibody Technique, Direct; Fluorescent Dyes; High-Throughput Screening Assays; Histones; Image Processing, Computer-Assisted; Male; Mice; Microscopy, Fluorescence; Neoplasm Proteins; Prostatic Neoplasms; Reproducibility of Results; Software; User-Computer Interface

Fragments of the extracellular matrix component hyaluronan (HA) promote tissue inflammation, fibrosis and tumor progression. HA fragments act through HA receptors including CD44, LYVE1, TLR2, 4 and the receptor for hyaluronan mediated motility (RHAMM/HMMR). RHAMM is a multifunctional protein with both intracellular and extracellular roles in cell motility and proliferation. Extracellular RHAMM binds directly to HA fragments while intracellular RHAMM binds directly to ERK1 and tubulin. Both HA and regions of tubulin (s-tubulin) are anionic and bind to basic amino acid-rich regions in partner proteins, such as in HA and tubulin binding regions of RHAMM. We used this as a rationale for developing bioinformatics and SPR (surface plasmon resonance) based screening to identify high affinity anionic RHAMM peptide ligands. A library of 12-mer peptides was prepared based on the carboxyl terminal tail sequence of s-tubulin isoforms and assayed for their ability to bind to the HA/tubulin binding region of recombinant RHAMM using SPR. This approach resulted in the isolation of three 12-mer peptides with nanomolar affinity for RHAMM. These peptides bound selectively to RHAMM but not to CD44 or TLR2,4 and blocked RHAMM:HA interactions. Furthermore, fluorescein-peptide uptake by PC3MLN4 prostate cancer cells was blocked by RHAMM mAb but not by CD44 mAb. These peptides also reduced the ability of prostate cancer cells to degrade collagen type I. The selectivity of these novel HA peptide mimics for RHAMM suggest their potential for development as HA mimetic imaging and therapeutic agents for HA-promoted disease.

Topics: Amino Acid Sequence; Biomimetic Materials; Breast Neoplasms; Carbocyanines; Cell Line, Tumor; Cell Movement; Drug Evaluation, Preclinical; Enzyme-Linked Immunosorbent Assay; Female; Fluorescent Dyes; Humans; Hyaluronan Receptors; Hyaluronic Acid; Ligands; Male; Molecular Sequence Data; Neoplasm Invasiveness; Peptide Library; Prostatic Neoplasms; Surface Plasmon Resonance; Tubulin

To synthesize and evaluate a peptide targeted nanoglobular dual modal imaging agent specific to a cancer biomarker in tumor stroma for MRI and fluorescence visualization of prostate tumor in image-guided surgery.. A peptide (CGLIIQKNEC, CLT1) targeted generation 2 nanoglobular (polylysine dendrimer with a silsesquioxane core) dual modal imaging agent, CLT1-G2-(Gd-DOTA-MA)-Cy5, was synthesized by stepwise conjugation of Gd-DOTA-MA, Cy5 and peptide to the dendrimer. Contrast enhanced MR imaging of the targeted dual imaging agent was evaluated on a Bruker 7T animal scanner with male athymic nude mice bearing orthotopic PC3-GFP prostate tumor. Fluorescence tumor imaging of the agent was carried out on a Maestro fluorescence imaging system.. The targeted agent CLT1-G2-(Gd-DOTA-MA)-Cy5 produced greater contrast enhancement in the tumor tissue than the control agent KAREC-G2-(Gd-DOTA-MA)-Cy5 at a dose of 30 μmol-Gd/kg in the MR images of the tumor bearing mice. Signal-to-noise ratio (SNR) of CLT1-G2-(Gd-DOTA-MA)-Cy5 in the tumor tissue was approximately 2 fold of that of the control agent in the first 15 min post-injection. The targeted agent also resulted in bright fluorescence signals in the tumor tissue.. The CLT1 peptide targeted nanoglobular dual-imaging agent CLT1-G2-(Gd-DOTA-MA)-Cy5 has a potential for MRI and fluorescence visualization of prostate tumor.

Topics: Animals; Carbocyanines; Cell Line, Tumor; Contrast Media; Fluorescence; Fluorescent Dyes; Heterocyclic Compounds; Humans; Magnetic Resonance Imaging; Male; Mice; Mice, Nude; Organometallic Compounds; Prostatic Neoplasms; Surgery, Computer-Assisted

Tumor cells are inherently heterogeneous and often exhibit diminished adhesion, resulting in the shedding of tumor cells into the circulation to form circulating tumor cells (CTCs). A fraction of these are live CTCs with potential of metastatic colonization whereas others are at various stages of apoptosis making them likely to be less relevant to understanding the disease. Isolation and characterization of live CTCs may augment information yielded by standard enumeration to help physicians to more accurately establish diagnosis, choose therapy, monitor response, and provide prognosis. We previously reported on a group of near-infrared (NIR) heptamethine carbocyanine dyes that are specifically and actively transported into live cancer cells. In this study, this viable tumor cell-specific behavior was utilized to detect live CTCs in prostate cancer patients. Peripheral blood mononuclear cells (PBMCs) from 40 patients with localized prostate cancer together with 5 patients with metastatic disease were stained with IR-783, the prototype heptamethine cyanine dye. Stained cells were subjected to flow cytometric analysis to identify live (NIR(+)) CTCs from the pool of total CTCs, which were identified by EpCAM staining. In patients with localized tumor, live CTC counts corresponded with total CTC numbers. Higher live CTC counts were seen in patients with larger tumors and those with more aggressive pathologic features including positive margins and/or lymph node invasion. Even higher CTC numbers (live and total) were detected in patients with metastatic disease. Live CTC counts declined when patients were receiving effective treatments, and conversely the counts tended to rise at the time of disease progression. Our study demonstrates the feasibility of applying of this staining technique to identify live CTCs, creating an opportunity for further molecular interrogation of a more biologically relevant CTC population.

Topics: Calibration; Carbocyanines; Cell Count; Cell Line, Tumor; Cell Separation; Coloring Agents; Disease Progression; Humans; Infrared Rays; Male; Neoplasm Metastasis; Neoplastic Cells, Circulating; Prostatectomy; Prostatic Neoplasms

Near-infrared (NIR) fluorescence imaging agents are promising tools for noninvasive cancer imaging. This study explored the specific uptake and retention of a NIR heptamethine carbocyanine MHI-148 dye by canine cancer cells and tissues and human prostate cancer (PCa) specimens and also the dye uptake mechanisms. The accumulation of MHI-148 was detected specifically in canine cancer cells and tissues and freshly harvested human PCa tissues xenografted in mice by NIR fluorescence microscopy and whole-body NIR optical imaging. Specific dye uptake in canine spontaneous tumors was further confirmed by PET imaging. Higher hypoxia-inducible factor-1α (HIF-1α) and organic anion-transporting polypeptide (OATP) protein and mRNA expression was demonstrated by multiplex quantum dots labeling and qPCR in tumors over that of normal tissues. Treating cancer cells with HIF-1α stabilizers activated HIF-1α downstream target genes, induced OATP superfamily gene expression and enhanced cellular uptake and retention of NIR dyes. Moreover, silencing HIF-1α by siRNA significantly decreased OATP mRNA expression and blocked NIR dye uptake in cancer cells. Together, these results demonstrated the preferential uptake of NIR dyes by canine and human cancer cells and tissues via the HIF-1α/OATPs signaling axis, which provides insights into future application of these dyes for cancer detection and treatment.

Topics: Animals; Carbocyanines; Cell Line, Tumor; Diagnostic Imaging; Dogs; Fluorescent Dyes; Gene Expression Regulation, Neoplastic; HEK293 Cells; Heterografts; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Male; Mice; Mice, Nude; Organic Anion Transporters; Prostatic Neoplasms; Signal Transduction; Spectroscopy, Near-Infrared

To study the effectiveness of a peptide targeted nanoglobular Gd-DOTA complexes for MR molecular imaging of prostate cancer in a mouse orthotopic PC-3 prostate cancer model.. A CLT1 (CGLIIQKNEC) peptide-targeted generation 2 nanoglobular Gd-DOTA monoamide conjugate [CLT1-G2-(Gd-DOTA)] was used for imaging fibrin-fibronectin complexes in prostate tumor using a non-specific peptide KAREC modified conjugate, KAREC-G2-(Gd-DOTA) as a control. Cy5 conjugates of CLT1 and KAREC were synthesized for binding studies. Orthotopic PC-3 prostate tumors were established in the prostate of athymic male nude mice. MRI study was performed on a Bruker 7T small animal MRI system.. CLT1 peptide showed specific binding in the prostate tumor with no binding in normal tissues. The control peptide had little binding in normal and tumor tissues. CLT1-G2-(Gd-DOTA) resulted in stronger contrast enhancement in tumor tissue than KAREC-G2-(Gd-DOTA). CLT1-G2-(Gd-DOTA) generated ~100% increase in contrast-to-noise ratio (CNR) in the tumor compared to precontrast CNR at 1 min post-injection, while KAREC-G2-(Gd-DOTA) resulted in 8% increase.. CLT1-G2-(Gd-DOTA) is a promising molecular MRI contrast agent for fibrin-fibronectin complexes in tumor stroma. It has potential for diagnosis and assessing prognosis of malignant tumors with MRI.

Topics: Animals; Carbocyanines; Cell Line, Tumor; Contrast Media; Disease Models, Animal; Fibrin; Fibronectins; Fluorescent Dyes; Heterocyclic Compounds; Humans; Magnetic Resonance Imaging; Male; Mice; Mice, Nude; Molecular Imaging; Nanoparticles; Organometallic Compounds; Peptides, Cyclic; Prostatic Neoplasms; Transplantation, Heterologous

We investigated the importance of HMGN5, a nuclear protein that binds to nucleosomes, unfolds chromatin, and affects transcription, in the LNCaP prostate cancer cell line. We also examined the molecular mechanisms that promote apoptosis of LNCaP cells after infection with small interfering RNA (siRNA) targeting HMGN5 (siRNA-HMGN5). The androgen-dependent LNCaP human prostate cancer cells were infected with siRNA-HMGN5. Apoptosis was detected using the Annexin V-PE/7-AAD double staining and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assays. Mitochondrial membrane potential was measured by JC-1 staining. HMGN5 and GAPDH mRNA expression were determined using real-time PCR. Bcl-2 and other apoptosis-related protein levels were determined by Western blot analysis. Caspase activity was measured by cleavage of the caspase substrate. Infection with siRNA targeting HMGN5 efficiently and specifically reduced the HMGN5 expression in LNCaP cells. The downregulation of HMGN5 induced remarkable apoptosis of LNCaP cells and resulted in the reduction of mitochondrial membrane potential. The induction of cell apoptosis was accompanied by the upregulation of Bax, the Bax/Bcl-2 ratio and the activation of caspase3. The HMGN5-targeted siRNA was effective in downregulating the expression of HMGN5 in androgen-dependent prostate cancer cells and inducing cell apoptosis via the regulation of a caspase-related mitochondrial pathway and Bcl-2 family proteins. This study suggests that HMGN5 may be a potential molecular target with therapeutic relevance for the treatment of prostate cancer.

Topics: Apoptosis; Benzimidazoles; Carbocyanines; Cell Line, Tumor; Cell Survival; Fluorescent Dyes; Gene Expression Regulation, Neoplastic; Gene Silencing; Genetic Therapy; HMGN Proteins; Humans; Male; Membrane Potential, Mitochondrial; Mitochondrial Membranes; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Real-Time Polymerase Chain Reaction; RNA Interference; RNA, Small Interfering; Trans-Activators

Prostate-specific membrane antigen (PSMA), a type II membrane glycoprotein, its high expression is associated with prostate cancer progression, and has been becoming an active target for imaging or therapeutic applications for prostate cancer. On the other hand, streptavidin-biotin system has been successfully employed in pretargeting therapy towards multiple cancers. Herein, we describe the synthesis of bifunctional ligands (biotin-CTT54, biotin-PEG(4)-CTT54, and biotin-PEG(12)-CTT54) possessing two functional motifs separated by a length-varied polyethylene glycol (PEG) spacer: one (CTT54) binds tumor-marker PSMA and the other (biotin) binds streptavidin or avidin. All three compounds exhibited high potencies (IC(50) values: 1.21, 2.53, and 10nM, respectively) and irreversibility; but only biotin-PEG(12)-CTT54 demonstrated specifically labeling PSMA-positive prostate cancer cells in a two-step pretargeting procedure. Additionally, the pre-formulated complex between biotin-PEG(12)-CTT54 and Cy5-streptavidin displayed the improved inhibitory potency (IC(50)=1.86 nM) and irreversibility against PSMA and rapid uptake of streptavidin conjugate into PSMA-positive prostate cancer cells through PSMA-associated internalization. Together, all these results supported a proof-concept that combination of streptavidin and PSMA's biotinylated inhibitor may lead to development of a novel strategy of tumor-targeting imaging or drug delivery towards prostate cancer.

Topics: Antibodies, Monoclonal; Antigens, Surface; Avidin; Biotin; Biotinylation; Carbocyanines; Cell Line, Tumor; Cell Survival; Drug Delivery Systems; Endocytosis; Enzyme Inhibitors; Fluorescence; Fluorescent Dyes; Glutamate Carboxypeptidase II; Humans; Immunoconjugates; Inhibitory Concentration 50; Male; Microscopy, Fluorescence; Organophosphorus Compounds; Polyethylene Glycols; Prostatic Neoplasms; Streptavidin

TIMP-2 has been studied as an attractive cancer therapeutic candidate, and a TIMP-2 fusion protein (HSA/TIMP-2) displayed effective anticancer activity, despite a lack of information about its pharmacokinetics (PK) and biodistribution. The purpose of this work was to assess the PK and biodistribution of HSA/TIMP-2 as well as to quantify accumulated HSA/TIMP-2 in tumors.. Cy5.5 near-infrared (NIR) fluorescence was conjugated to the HSA/TIMP-2 protein (Cy5.5-HSA/TIMP-2) for monitoring spatio-temporal changes in vivo. For PK and biodistribution analysis, 0.2 μg/g body weight of Cy5.5-HSA/TIMP-2 was injected into MAT-LyLu prostate tumor xenografts, which were then imaged using an IVIS-200 optical imaging system. To quantify the accumulated HSA/TIMP-2 in tumors, we introduced a standard curve with depth-corrected fluorescence measurement.. In the vascular tube formation assay with human umbilical vein endothelial cells (HUVECs), Cy5.5-HSA/TIMP-2 showed an antiangiogenic effect. In prostate cancer xenografts, Cy5.5-HSA/TIMP-2 exhibited a prolongation of blood half-life to 19.6 h and relatively preferential distribution to the tumor. The amount of tumor-accumulated Cy5.5-HSA/TIMP-2 was calculated to be 4.5 ± 0.5 ng/g body weight at 2 days, representing 2.25 ± 0.25% of the initial dose.. We evaluated the pharmacokinetic profile and biodistribution of HSA/TIMP-2 with favorable results, providing new information for more effective approaches to cancer therapeutics using HSA/TIMP-2. Additionally, real-time in vivo fluorescence imaging analysis using a depth-corrected standard curve may serve as a platform to quantify biodistributed drug in anticancer therapeutic studies.

Topics: Angiogenesis Inhibitors; Animals; Carbocyanines; Half-Life; Human Umbilical Vein Endothelial Cells; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; Prostatic Neoplasms; Rats; Recombinant Fusion Proteins; Serum Albumin; Serum Albumin, Human; Spectroscopy, Near-Infrared; Tissue Distribution; Tissue Inhibitor of Metalloproteinase-2; Transplantation, Heterologous; Tumor Cells, Cultured

Prostate-specific membrane antigen (PSMA) remains an active target for imaging and therapeutic applications for prostate cancer. Although radionuclide-based imaging is generally more sensitive and also has been deeply explored, near-infrared fluorescence imaging agents are simple to prepare and compatible with long-term storage conditions. In the present study, a near-infrared fluorescent imaging probe (Cy5.5-CTT-54.2) has been developed by chemical conjugation of Cy5.5N-hydroxysuccinimide ester (Cy5.5-NHS) with a potent PSMA inhibitor CTT-54.2 (IC(50)=144 nM). The probe displays a highly potency (IC(50)=0.55 nM) against PSMA and has demonstrated successful application for specifically labeling PSMA-positive prostate cancer cells in both two and three-dimensional cell culture conditions. These results suggest that the potent, near-infrared Cy5.5-PSMA inhibitor conjugate may be useful for the detection of prostate tumor cells by optical in vivo imaging.

Topics: Carbocyanines; Fluorescent Dyes; Humans; Infrared Rays; Male; Molecular Weight; Prostate-Specific Antigen; Prostatic Neoplasms; Tumor Cells, Cultured

B cell lymphoma 2 (Bcl-2) homology domain 3 (BH3)-only proteins of the Bcl-2 family are important functional adaptors that link cell death signals to the activation of Bax and/or Bak. The BH3-only protein Nbk/Bik induces cell death via an entirely Bax-dependent/Bak-independent mechanism. In contrast, cell death induced by the short splice variant of Bcl-x depends on Bak but not Bax. This indicates that Bak is functional but fails to become activated by Nbk. Here, we show that binding of myeloid cell leukemia 1 (Mcl-1) to Bak persists after Nbk expression and inhibits Nbk-induced apoptosis in Bax-deficient cells. In contrast, the BH3-only protein Puma disrupts Mcl-1-Bak interaction and triggers cell death via both Bax and Bak. Targeted knockdown of Mcl-1 overcomes inhibition of Bak and allows for Bak activation by Nbk. Thus, Nbk is held in check by Mcl-1 that interferes with activation of Bak. The finding that different BH3-only proteins rely specifically on Bax, Bak, or both has important implications for the design of anticancer drugs targeting Bcl-2.

Topics: Adenoviridae; Apoptosis; Apoptosis Regulatory Proteins; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Benzimidazoles; Carbocyanines; Cell Line; Cell Line, Tumor; Cell Membrane Permeability; Cytochromes c; Fluorescent Dyes; HCT116 Cells; Humans; Kidney; Male; Membrane Proteins; Mitochondria; Mitochondrial Proteins; Models, Biological; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Proteins; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Transgenes

Spotted cDNA microarrays generally employ co-hybridization of fluorescently-labeled RNA targets to produce gene expression ratios for subsequent analysis. Direct comparison of two RNA samples in the same microarray provides the highest level of accuracy; however, due to the number of combinatorial pair-wise comparisons, the direct method is impractical for studies including large number of individual samples (e.g., tumor classification studies). For such studies, indirect comparisons using a common reference standard have been the preferred method. Here we evaluated the precision and accuracy of reconstructed ratios from three indirect methods relative to ratios obtained from direct hybridizations, herein considered as the gold-standard.. We performed hybridizations using a fixed amount of Cy3-labeled reference oligonucleotide (RefOligo) against distinct Cy5-labeled targets from prostate, breast and kidney tumor samples. Reconstructed ratios between all tissue pairs were derived from ratios between each tissue sample and RefOligo. Reconstructed ratios were compared to (i) ratios obtained in parallel from direct pair-wise hybridizations of tissue samples, and to (ii) reconstructed ratios derived from hybridization of each tissue against a reference RNA pool (RefPool). To evaluate the effect of the external references, reconstructed ratios were also calculated directly from intensity values of single-channel (One-Color) measurements derived from tissue sample data collected in the RefOligo experiments. We show that the average coefficient of variation of ratios between intra- and inter-slide replicates derived from RefOligo, RefPool and One-Color were similar and 2 to 4-fold higher than ratios obtained in direct hybridizations. Correlation coefficients calculated for all three tissue comparisons were also similar. In addition, the performance of all indirect methods in terms of their robustness to identify genes deemed as differentially expressed based on direct hybridizations, as well as false-positive and false-negative rates, were found to be comparable.. RefOligo produces ratios as precise and accurate as ratios reconstructed from a RNA pool, thus representing a reliable alternative in reference-based hybridization experiments. In addition, One-Color measurements alone can reconstruct expression ratios without loss in precision or accuracy. We conclude that both methods are adequate options in large-scale projects where the amount of a common reference RNA pool is usually restrictive.

Topics: Adenocarcinoma; Breast Neoplasms; Carbocyanines; Carcinoma, Renal Cell; DNA, Complementary; DNA, Neoplasm; Female; Fluorescent Dyes; Gene Expression Regulation, Neoplastic; Humans; Kidney Neoplasms; Male; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; Prostatic Neoplasms

Diagnosing cancers based on serum profiling is a particularly attractive concept. However, the technical challenges to analysis of the serum proteome arise from the dynamic range of protein amounts. Cancer sera contain antibodies that react with a unique group of autologous cellular antigens, which affords a dramatic amplification of signal in the form of antibodies relative to the amount of the corresponding antigens. The serum autoantibody repertoire from cancer patients might, therefore, be exploited for antigen-antibody profiling. To date, studies of antigen-antibody reactivity using microarrays have relied on recombinant proteins or synthetic peptides as arrayed features. However, recombinant proteins and/or synthetic peptides may fail to accurately detect autoantibody binding due to the lack of proper PTMs. Here we describe the development and use of a "reverse capture" autoantibody microarray. Our "reverse capture" autoantibody microarray is based on the dual-antibody sandwich immunoassay platform of ELISA, which allows the antigens to be immobilized in their native configuration. As "proof-of-principle", we demonstrate its use for antigen-autoantibody profiling with sera from patients with prostate cancer and benign prostate hyperplasia.

Topics: Aged; Antibodies, Monoclonal; Antigens, Neoplasm; Autoantibodies; Biomarkers, Tumor; Blotting, Western; Carbocyanines; Cell Line; Enzyme-Linked Immunosorbent Assay; Fluorescent Dyes; Humans; Immunoprecipitation; Male; Middle Aged; Prostatic Hyperplasia; Prostatic Neoplasms; Protein Array Analysis; Serum

We designed a fluorescent peptide-magnetic nanoparticle conjugate that images E-selectin expression in mouse xenograft models of Lewis lung carcinoma (LLC) by fluorescence reflectance imaging. It was synthesized by attaching the E-selectin-binding peptide (ESBP; CDSDSDITWDQLWDLMK) to a CLIO(Cy5.5) nanoparticle to yield ESBP-CLIO(Cy5.5). Internalization by activated human umbilical vein endothelial cells (HUVECs) was rapid and mediated by E-selectin, indicated by the lack of uptake of nanoparticles bearing similar numbers of a scrambled peptide (Scram). To demonstrate the specificity of E-selectin targeting to ESBP-CLIO(Cy5.5) in vivo, we coinjected ESBP-CLIO(Cy5.5) and Scram-CLIO(Cy3.5) and demonstrated a high Cy5.5/Cy3.5 fluorescence ratio using the LLC. Histology showed that ESBP-CLIO was associated with tumor cells as well as endothelial cells, but fluorescence-activated cell sorter analysis showed a far less expression of E-selectin on LLC than on HUVECs. Using immunohistochemistry, we demonstrated E-selectin expression in both endothelial cells and cancer cells in human prostate cancer specimens. We conclude that ESBP-CLIO(Cy5.5) is a useful probe for imaging E-selectin associated with the LLC tumor, and that E-selectin is expressed not only on endothelial cells but also on LLC cells and human prostate cancer specimens.

Topics: Animals; Carbocyanines; Carcinoma, Lewis Lung; Cell Line; Cell Line, Tumor; Cell Nucleus; Cell Separation; E-Selectin; Edetic Acid; Endothelial Cells; Endothelium, Vascular; Flow Cytometry; Humans; Immunohistochemistry; Interleukin-1; Male; Mice; Microscopy, Confocal; Microscopy, Fluorescence; Nanostructures; Nanotechnology; Neoplasm Transplantation; Peptides; Platelet Endothelial Cell Adhesion Molecule-1; Prostatic Neoplasms; Sensitivity and Specificity; Substrate Specificity; Time Factors; Umbilical Veins