carbocyanines and Melanoma

In recent decades, zebrafish (Danio rerio) has become a major in vivo model for the evaluation of drug efficacies and toxicities. In the field of drug delivery research, zebrafish larvae are a suitable model for the use of fluorescent-labeled chemicals, nanoparticle, liposome, or micelle-mediated delivery systems because of their transparent body wall. In the current chapter, we describe the method to perform micelle-based siRNA delivery using cancer cells implanted into the circulation of zebrafish.

Topics: Animals; Animals, Genetically Modified; Carbocyanines; Cell Line, Tumor; Cell Transplantation; Drug Delivery Systems; Fluorescent Dyes; Gene Transfer Techniques; Humans; Larva; Luminescent Proteins; Melanoma; Micelles; Polylactic Acid-Polyglycolic Acid Copolymer; Red Fluorescent Protein; RNA, Small Interfering; Transplants; Zebrafish

A new type of photodynamic therapy (PDT) agents using upconversion nanoparticles (UCNPs) with incorporated photosensitizers as the inner core and an erythrocyte membrane (RM) decorated with dual targeting moieties as the cloak is developed. Owing to the endogenous nature of RM, the RM-coating endows the PDT agents with perfect biocompatibility and stealth ability to escape from the entrapment by the reticulo-endothelial system (RES). More importantly, owing to the unique nature of erythrocyte as an oxygen carrier in the blood, the RM outer layer of the agents unequivocally facilitates the permeation of ground-state molecular oxygen ((3)O2) and the singlet oxygen ((1)O2) as compared to the previously developed PDT agents with other types of coating. Another salient feature of the as-prepared PDT platform is the decoration of RM with dual targeting moieties for selective recognition of cancer cells and mitochondrial targeting, respectively. The synergistic effect of RM coating and dual-targeting of such feature-packed agents are investigated in tumor-bearing mice and the improved PDT therapeutic efficacy is confirmed, which is the first paradigm where RM-coated NIR-triggered nanovectors with programmed delivery ability is applied in PDT of tumor in vivo.

Topics: Animals; Biomimetic Materials; Carbocyanines; Cell Line, Tumor; Disease Models, Animal; Erythrocyte Membrane; Infrared Rays; Melanoma; Mice; Microscopy, Confocal; Nanoparticles; Photochemotherapy; Photosensitizing Agents; Singlet Oxygen; Skin Neoplasms; Spectroscopy, Near-Infrared; Survival Rate

On the basis of the fact of the overexpression of integrins in malignant tumor cells and neovasculature, and the advantage of polymeric micelles (PM) as the drug carriers, a cyclic RGD peptide (cRGDyK) was anchored on the surface of polyethylene glycol-b-poly(lactic-co-glycolic acid) (PEG-b-PLGA) micelles as a ligand of integrins in order to enhance the intracellular delivery of encapsulated hydrophobic drug into the tumor cells and its neovasculature. Toward this goal, PEG-b-PLGA micelles without or with cRGDyK conjugation loaded with paclitaxel (PTX) or DiI were prepared and characterized. The results revealed that drug-loaded micelles were stable in solution, with small diameters (<80 nm) and a low critical micelle concentration. Spectrophotofluorometry, confocal microscopy, and flow cytometry showed that cRGDyK-conjugated micelles (TPM) facilitated the cell-specific uptake of DiI into the murine melanoma B16-F10 cells and human umbilical vein endothelial cells (HUVEC) via integrin-mediated endocytosis compared with cRGDyK-free micelles (NPM), and the uptake was proportional to the ratio of cRGDyK modification in certain range. Meanwhile, PTX-loaded TPM displayed higher cytotoxicity and antiproliferation activities against both cells than PTX-loaded NPM. These results suggest that cRGDyK-coupled PEG-b-PLGA micelles may be the promising intracellular targeting carriers for efficient delivery of chemotherapeutic agents into tumor cells and neovasculature.

Topics: Animals; Carbocyanines; Cell Line, Tumor; Drug Carriers; Drug Delivery Systems; Endocytosis; Endothelial Cells; Flow Cytometry; Fluorescent Dyes; Humans; Integrins; Melanoma; Mice; Micelles; Microscopy, Confocal; Paclitaxel; Particle Size; Peptides, Cyclic; Polyethylene Glycols; Polyglactin 910

Difference in-gel electrophoresis (DIGE)-based protein expression analysis allows assessing the relative expression of proteins in two biological samples differently labeled (Cy5, Cy3 CyDyes). In the same gel, a reference sample is also used (Cy2 CyDye) for spot matching during image analysis and volume normalization. The standard statistical techniques to identify differentially expressed (DE) proteins are the calculation of fold-changes and the comparison of treatment means by the t-test. The analyses rarely accounts for other experimental effects, such as CyDye and gel effects, which could be important sources of noise while detecting treatment effects.. We propose to identify DIGE DE proteins using a two-stage linear mixed model. The proposal consists of splitting the overall model for the measured intensity into two interconnected models. First, we fit a normalization model that accounts for the general experimental effects, such as gel and CyDye effects as well as for the features of the associated random term distributions. Second, we fit a model that uses the residuals from the first step to account for differences between treatments in protein-by-protein basis. The modeling strategy was evaluated using data from a melanoma cell study. We found that a heteroskedastic model in the first stage, which also account for CyDye and gel effects, best normalized the data, while allowing for an efficient estimation of the treatment effects. The Cy2 reference channel was used as a covariate in the normalization model to avoid skewness of the residual distribution. Its inclusion improved the detection of DE proteins in the second stage.

Topics: Carbocyanines; Cell Line, Tumor; Computational Biology; Electrophoresis, Gel, Two-Dimensional; Fluorescent Dyes; Humans; Image Processing, Computer-Assisted; Linear Models; Melanoma; Proteome; Proteomics

The molecular changes involved in the induction of apoptosis by vincristine in melanoma have not yet been well defined. Two human melanoma cell lines showing moderate (Mel-RM) and high (IgR3) sensitivity to vincristine were selected from a panel of eight melanoma lines for analysis. Induction of apoptosis was caspase dependent, and was associated with increases in mitochondrial membrane permeability. Vincristine upregulated the expression of Bax, Bak, PUMA, Noxa, p53 and p21 proteins, and downregulated and/or phosphorylated the Bcl-2 protein. Inhibitors of the Jun N-terminal kinase (JNK), but not p38 mitogen-activated protein kinase, significantly inhibited vincristine-induced apoptosis in both IgR3 and Mel-RM cells. In addition, vincristine induced phosphorylation and reduction in Bcl-2 was prevented by an inhibitor of JNK. Downregulation of mRNA for p53, PUMA or Bim by RNA interference had little or no influence on vincristine-induced apoptosis in IgR3 cells. In addition, silencing Bim mRNA did not affect vincristine-induced apoptosis in Mel-RM cells. These results suggest that vincristine-induced apoptosis of at least some melanoma cell lines is dependent on the activation of JNK. The results are consistent with the phosphorylation of Bcl-2 protein, resulting in the activation of Bax/Bak, release of cytochrome c from the mitochondria and the resulting activation of caspases.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; Benzimidazoles; Blotting, Western; Carbocyanines; Caspases; Cell Line, Tumor; Dose-Response Relationship, Drug; Enzyme Activation; Flow Cytometry; Humans; JNK Mitogen-Activated Protein Kinases; Melanoma; Membrane Potential, Mitochondrial; Membrane Proteins; Mitochondrial Membrane Transport Proteins; Phosphorylation; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; RNA, Messenger; Signal Transduction; Tubulin Modulators; Tumor Suppressor Protein p53; Vincristine

Use of fluorescence imaging in oncology is evolving rapidly, and nontargeted fluorochromes are currently being investigated for clinical application. Here, we investigated whether the degree of tumour angiogenesis can be assessed in vivo by planar and tomographic methods using the perfusion-type cyanine dye SIDAG (1,1'-bis- [4-sulfobutyl]indotricarbocyanine-5,5'-dicarboxylic acid diglucamide monosodium).. Mice were xenografted with moderately (MCF7, DU4475) or highly vascularized (HT1080, MDA-MB435) tumours and scanned up to 24 hours after intravenous SIDAG injection using fluorescence reflectance imaging. Contrast-to-noise ratio was calculated for all tumours, and fluorochrome accumulation was quantified using fluorescence-mediated tomography. The vascular volume fraction of the xenografts, serving as a surrogate marker for angiogenesis, was measured using magnetic resonance imaging, and blood vessel profile (BVP) density and vascular endothelial growth factor expression were determined.. SIDAG accumulation correlated well with angiogenic burden, with maximum contrast to noise ratio for MDA-MB435 (P < 0.0001), followed by HT1080, MCF7 and DU4475 tumours. Fluorescence-mediated tomography revealed 4.6-fold higher fluorochrome concentrations in MDA-MB435 than in DU4475 tumours (229 +/- 90 nmol/l versus 49 +/- 22 nmol/l; P < 0.05). The vascular volume fraction was 4.5-fold (3.58 +/- 0.9% versus 0.8 +/- 0.53%; P < 0.01), blood vessel profile density 5-fold (399 +/- 36 BVPs/mm2 versus 78 +/- 16 BVPs/mm2) and vascular endothelial growth factor expression 4-fold higher for MDA-MB435 than for DU4475 tumours.. Our data suggest that perfusion-type cyanine dyes allow assessment of angiogenesis in vivo using planar or tomographic imaging technology. They may thus facilitate characterization of solid tumours.

Topics: Adenocarcinoma; Animals; Blotting, Western; Breast Neoplasms; Carbocyanines; Cell Line, Tumor; Contrast Media; Fibrosarcoma; Fluorescence; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Magnetic Resonance Imaging; Melanoma; Mice; Neoplasms; Neovascularization, Pathologic; Optics and Photonics; Tomography; Transplantation, Heterologous; Vascular Endothelial Growth Factor A

The breast cancer metastasis suppressor 1 (BRMS1) gene has been shown to suppress metastasis without affecting the growth of the primary tumor in mouse models. It has also been shown to suppress the metastasis of tumors derived from breast, melanoma, and, more recently, ovarian carcinoma (see ref 1). However, how BRMS1 exerts its metastasis suppressor function remains unknown. To shed light into its metastatic mechanism of action, the sensitive 2D-DIGE analysis coupled with MS has been used to identify proteins differentially expressed by either overexpressing (Mel-BRMS1) or silencing BRMS1 (sh635) in a melanoma cell line. After comparison of the protein profiles from WT, Mel-BRMS1, and sh635 cells, 79 spots were found to be differentially expressed. Mass spectrometry analysis allowed the unambiguous identification of 55 polypeptides, corresponding to 43 different proteins. Interestingly, more than 75% of the identified proteins were down-regulated in Mel-BRMS1 cells compared to WT. In contrast, all the identified proteins in sh635 cells extracts were up-regulated compared to WT. Most of the deregulated proteins are involved in cell growth/maintenance and signal transduction among other cell processes. Six differentially expressed proteins (Hsp27, Alpha1 protease inhibitor, Cofilin1, Cathepsin D, Bone morphogenetic protein receptor2, and Annexin2) were confirmed by immunoblot and functional assays. Excellent correlation was found between DIGE analysis and immunoblot results, indicating the reliability of the analysis. Available evidence on the reported functions of the identified proteins supports the emerging role of BRMS1 as negative regulator of the metastasis development. This work opens an avenue for the molecular mechanisms' characterization of metastasis suppressor genes with the aim to understand their roles.

Topics: Bone Morphogenetic Protein Receptors, Type II; Carbocyanines; Cathepsin D; Cell Line; Cell Line, Tumor; Data Interpretation, Statistical; Electrophoresis, Gel, Two-Dimensional; Fluorescent Dyes; Humans; Melanoma; Neoplasm Metastasis; Neoplasm Proteins; Proteome; Proteomics; Repressor Proteins; RNA Interference; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Choroidal melanoma is the most common primary ocular cancer among the adult population. To avoid enucleation, there has been a concerted effort to develop therapies that spare the affected eye and the patient's vision. Blood flow helps shape the tumor's microenvironment, plays a key role in the tumor's response to many different types of therapy, and is necessary for delivery of chemotherapeutic agents. To rationally design new therapies and optimize existing treatments, it is essential to learn as much as possible about blood flow and the microcirculation in this tumor. In recent years, much has been discovered about the anatomy of the microvasculature and the dynamics of overall blood flow in choroidal melanoma, but little is known about the factors that determine microvascular blood flow. In this study hemodynamic parameters in individual microvessels of a human choroidal melanoma xenograft were compared with those same parameters in a normal rat choroid.. Nude, athymic WAG/RijHs-rnu rats were used in this study. The human choroidal melanoma cell line OCM-1 was used to grow solid tumors subcutaneously in the flanks of donor rats. Small pieces of these tumors were then implanted into the choroidal space of recipient rats. After 6 to 8 weeks, the rats were anesthetized with a subcutaneous injection of urethane, and the sclera was exposed. Rhodamine-labeled liposomes and red blood cells (RBCs) labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) were injected intravenously. Epifluorescent, intravital microscopy was used to visualize the flow of fluorescent RBCs through individual vessels in the choroid or tumor. Flow through multiple vessels was recorded on videotape for later analysis. From the recordings, RBC flux, RBC velocity (V(c)), and microvascular hematocrit (HCT(m)) were determined. Similar experiments were performed in rats with no tumor growth, and these same parameters were calculated in normal choroidal vessels. RBC flow was characterized in 55 vessels in six OCM-1 tumors and in 153 choroidal vessels in five non-tumor-bearing rats.. RBC flux was higher in larger tumor vessels (>30 micro m in diameter) compared with similarly sized choroidal vessels. There were no differences in the velocities of RBCs through the two types of vessels. HCT(m) was significantly higher in medium-sized (>20 micro m in diameter) and larger tumor vessels compared with normal choroidal vessels.. These experiments demonstrate differences between hemodynamic parameters in normal choroidal microvessels and microvessels in choroidal melanoma in this animal model. Because HCT(m) is a key determinant of apparent viscosity, abnormally high HCT(m) in the tumor vessels would increase vascular resistance and decrease flow. This could have a negative impact on the tumor oxygen levels and on the ability to deliver drugs effectively. On the contrary, higher local HCT(m) has also been shown to increase oxygen delivery. The impact and interplay of these two effects on tumor oxygenation remain to be determined.

Topics: Animals; Blood Flow Velocity; Carbocyanines; Choroid; Choroid Neoplasms; Erythrocytes; Fluorescent Dyes; Hematocrit; Hemodynamics; Melanoma; Microcirculation; Microscopy, Fluorescence; Models, Animal; Neovascularization, Pathologic; Rats; Rats, Mutant Strains; Rhodamines; Transplantation, Heterologous; Video Recording

Far-red-emitting cyanine fluorochromes have many properties desirable for in vivo imaging: absorption and emission at wavelengths where blood and tissue are relatively transparent, high quantum yields, and good solubility even at high molar ratios of fluorochrome to antibody. Potentially, conjugation by multiple linkages should minimize hydrolysis in vivo. We conjugated two tumor-targeting monoclonal antibodies: anti-SSEA-1 (IgM, kappa) at ratios of 1.2-35 mol dye/mol antibody and 9.2.27 (IgG2a, kappa) at 0.6-6 mol dye/mol antibody, using the cyanine fluorochromes Cy3.18, Cy5.18, and Cy5.5.18. Nude mice were inoculated using the SSEA-1-expressing MH-15 teratocarcinoma or the 9.2.27 antigen-expressing SK-MEL-2 melanoma to give tumors at several sites. Conjugated antibody was injected, and mice were imaged immediately after injection and at appropriate intervals thereafter using a standard camera lens, dissecting microscope, or endoscopes. Images were acquired using either an image-intensified video camera or cooled CCD cameras. Immediately after injection, major blood vessels and the heart, liver, and kidneys were readily visualized. After 1 day, tumor-targeting antibody conjugates were concentrated in tumors and there was little circulating conjugate; however, the bladder and kidneys were still visible. Tumors labeled by specific antibody were the most fluorescent tissues at 2 days after injection, but non-specific antibody conjugates did not concentrate in the tumors. The small intestine was weakly visualized by both specific and non-specific antibody conjugates. These data support the possibility of visualizing tumor metastasis by optical means, including currently available endoscopes.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Neoplasm; Antigens, Neoplasm; Carbocyanines; Diagnostic Imaging; Fluorescent Antibody Technique; Humans; Melanoma; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasms, Experimental; Teratocarcinoma

Studies of transplantable rodent tumours have suggested that malignant tissue might experience transient perfusion at the microvascular level. The purpose of the work reported here was to investigate whether transient perfusion can be demonstrated in xenografted human tumours. Tumours of four melanoma lines (A-07, D-12, R-18, U-25), grown orthotopically in Balb/c nu/nu mice, were included in the study. Transient perfusion was studied by using the double-fluorescent staining technique. Hoechst 33342 and DiOC7(3) were either administered simultaneously or Hoechst 33342 was administered 20 min before DiOC7(3). Detection of transient perfusion by this method requires that vessels are non-functional for at least 5 min owing to the distribution half-lives of the dyes in the blood. Usable combinations of dye concentrations were found by varying the concentrations of Hoechst 33342 and DiOC7(3) systematically. The level of perfusion mismatch following simultaneous administration of the dyes ranged from approximately 1.5% for U-25 tumours to approximately 3.0% for R-18 tumours at these combinations. Moreover, the fraction of vessels stained only with Hoechst 33342 and the fraction of vessels stained only with DiOC7(3) were not significantly different whether the dyes were administered simultaneously or sequentially. Transient perfusion could not be demonstrated in any of the tumour lines. Thus, the fraction of vessels stained only with Hoechst 33342 and the fraction of vessels stained only with DiOC7(3) were not significantly higher after sequential than after simultaneous administration of the dyes. Moreover, the vessels stained only with Hoechst 33342 and the vessels stained only with DiOC7(3) were randomly distributed within the tumours whether the dyes were administered simultaneously or sequentially. Consequently, acute hypoxia caused by transient perfusion is probably a less pronounced phenomenon in malignant tissue than previous studies of rodent tumours have suggested.

Topics: Animals; Benzimidazoles; Carbocyanines; Cell Line; Fluorescent Dyes; Humans; Melanoma; Mice; Mice, Nude; Microcirculation; Perfusion; Staining and Labeling; Transplantation, Heterologous; Tumor Cells, Cultured

The search for improved photosensitizers for laser phototherapy of malignancies has led to the examination of a new group of carbocyanine dyes as effective fluorochromes. In this study, four carbocyanine dyes with different absorption maxima of 483 nm [DiOC6(3)], 545.5 nm (DiIC5(3)], 556.6 nm [DiSC5(3)], and 651.0 nm [DiSC3(5)] were tested in vitro. The kinetics of uptake and toxicity of these four dyes were assessed for P3 human squamous cell carcinoma, HT29 colon carcinoma, M26 melanoma, and TE671 fibrosarcoma cell lines at 15, 30, 45, 60, and 180 minutes after exposure with each dye. After sensitization with DiOC6(3), the P3 and M26 cell lines were also tested for phototherapy by treatment with 488-nm light from an argon laser. The results showed that these four carbocyanine dyes had rapid and significant uptake by the carcinoma cell lines with no toxicity at concentrations < 0.1 micrograms/mL. Nontoxic DiOC6(3) levels in sensitized tumor cells after laser phototherapy resulted in approximately 85% inhibition of P3 and approximately 95% inhibition of M26 cell lines by MTT assays. The results suggest that these carbocyanine dyes can be used for tumor photosensitization and wavelength-matched laser photodynamic therapy. Further in vivo studies will be necessary to define the clinical potential of carbocyanine dyes as tumor-targeting agents for phototherapy of cancer.

Topics: Adenocarcinoma; Argon; Benzothiazoles; Carbocyanines; Carcinoma, Squamous Cell; Cell Survival; Colonic Neoplasms; Fibrosarcoma; Fluorescent Dyes; Humans; Laser Therapy; Lung Neoplasms; Medulloblastoma; Melanoma; Neoplasms; Photochemotherapy; Tetrazolium Salts; Tumor Cells, Cultured