TRAIL-based tumor sensitizing galactoxyloglucan,a novel entity for targeting apoptotic machinery

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an attractive target for cancer therapy due to its ability to selectively induce apoptosis in cancer cells, without causing significant toxicity in normal tissues. We previously reported that galactoxyloglucan (PST001) possesses significant antitumor and immunomodulatory properties. However, the exact mechanism in mediating this anticancer effect is unknown. This study, for the first time, indicated that PST001 sensitizes non-small cell lung cancer (A549) and nasopharyngeal (KB) cells to TRAIL-mediated apoptosis. In vitro studies suggested that PST001 induced apoptosis primarily via death receptors and predominantly activated caspases belonging to the extrinsic apoptotic cascade. Microarray profiling of PST001 treated A549 and KB cells showed the suppression of survivin (BIRC5) and anti-apoptotic Bcl-2, as well as increased cytochrome C. TaqMan low density array analysis of A549 cells also confirmed that the induction of apoptosis by the polysaccharide occurred through the TRAIL-DR4/DR5 pathways. This was finally confirmed by in silico analysis, which revealed that PST001 binds to TRAIL-DR4/DR5 complexes more strongly than TNF and Fas ligand–receptor complexes. In summary, our results suggest the potential of PST001 to be developed as an anticancer agent that not only preserves innate biological activity of TRAIL, but also sensitizes cancer cells to TRAIL-mediated apoptosis.     

Canine transmissible venereal tumor: a large-animal transplantable tumor model

The canine transplantable venereal tumor is a naturally occurring transplantable round-cell tumor in dogs. Although experimental transplantable tumor models in rodents and rabbits are readily available, a reliable transplantable tumor model in a large animal that more closely resembles the physical dimensions of humans has not been available. A tumor model in a large animal would have a wide range of biomedical research applications, including the study of various interventional imaging techniques. In this report, we characterize the experimental transplantation of the canine transmissible venereal tumor in the brain, skin, muscle, prostate, lung, liver, and bone of dogs and provide X-ray computed tomographic and magnetic resonance imaging characteristics of the tumors in the brain, muscle, lung, and prostate.     

Synthesis and exploration of novel radiolabeled bombesin peptides for targeting receptor positive tumor

Increasing evidence of peptide receptor overexpression in various cancer cells, warrant the development of receptor specific radiolabeled peptides for molecular imaging and therapy in nuclear medicine. Gastrin-releasing-peptide (GRP) receptor, are overexpressed in a variety of human cancer cells. The present study report the synthesis and biological evaluation of new bombesin (BBN) analogs, HYNIC-Asp-[Phe¹³]BBN(7–13)-NH-CH₂-CH₂-CH3:BA1, HYNIC-Pro-[Tyr¹³Met¹⁴]BBN(7–14)NH₂:BA2 as prospective tumor imaging agent with compare to BBN(7–14)NH₂:BS as standard. The pharmacophores were radiolabeled in high yields with ^99mTc, characterized for their stability in serum and saline, cysteine/histidine and were found to be substantially stable. Internalization/externalization and receptor binding studies were assessed using MDA-MB-231 cells and showed high receptor binding-affinity and favourable internalization. Fluorescence studies revealed that BA1 changed the morphology of the cells and could localize in the nucleus more effectively than BA2/BS. Cell-viability studies displayed substantial antagonistic and nuclear-internalization effect of BA1. BA1 also exhibited antiproliferative effect on MDA-MB-231 cell by inducing apoptosis. In vivo behaviour of the radiopeptides was evaluated in GRP receptor positive tumor bearing mice. The ^99mTc-BA1/^99mTc-BA2 demonstrated rapid blood/urinary clearance through the renal pathway and comparatively more significant tumor uptake image and favourable tumor-to-non-target ratios provided by ^99mTc-BA1. The specificity of the in vivo uptake was confirmed by co-injection with BS. Moreover, ^99mTc-BA1 provided a much clearer tumor image in scintigraphic studies than others. Thus the combination of favourable in vitro and in vivo properties renders BA1 as more potential antagonist bombesin-peptide for targeting GRP-receptor positive tumor. These properties are encouraging to carry out further experiments for non-invasive receptor targeting potential diagnostinc and therapeutic agent for tumors.     

Avicins, a novel plant-derived metabolite lowers energy metabolism in tumor cells by targeting the outer mitochondrial membrane

Avicins are pro-apoptotic, anti-inflammatory molecules with antioxidant effects both in vitro and in vivo. Based on their ability to perturb mitochondrial functions and initiate apoptosis in tumor cells, we chose to study the bioenergetic effects of avicins on tumor cell mitochondria. Avicin-treated Jurkat cells, showed a decrease in the levels of cellular ATP as well as the rate of oxygen consumption. These effects on cellular metabolism appear to be a result of avicin's actions on the outer mitochondrial membrane (OMM). We speculate that avicins might initially inhibit the exchange of metabolites across the OMM leading to its subsequent permeabilization to cytochrome c. This speculation is supported by biophysical studies using lipid bilayers, which suggest that upstream of these effects, avicins target and close the voltage dependent anion channel (VDAC). Closure of VDAC would lead to an overall lowering of the cell energy metabolism, subsequently pushing these cells towards the apoptotic pathway by permeabilization of the OMM and release of cyt-c. Avicins therefore not only represent a novel pharmacological tool for treatment of cancers, but also highlight the influence ancient plant metabolites could have on human health.     

A novel, non-immunogenic Fyn SH3-derived binding protein with tumor vascular targeting properties

The generation of novel binding molecules based on protein frameworks ("scaffolds") represents an emerging field in protein engineering, with the potential to replace antibodies for many research and clinical applications. Here, we describe the design, construction, characterization, and use of a novel human Fyn SH3 phage library, containing 1.2 x 10(9) individual clone members. We also present the isolation and in vitro characterization of Fyn SH3-derived proteins binding to the extra-domain B of fibronectin, a marker of angiogenesis. One specific binding clone, named D3, was further evaluated and showed a remarkable ability to stain vascular structures in tumor sections. Furthermore, quantitative biodistribution studies in tumor-bearing mice revealed the ability of D3 to selectively accumulate in the tumor. In contrast to human scFv antibody fragments administered to mice, neither Fyn SH3 WT nor the D3 mutant was immunogenic in mice after four intravenous injections. The extra-domain B binding D3 protein opens new biomedical opportunities for the in vivo imaging of solid tumors and for the delivery of toxic agents to the tumoral vasculature.     

Antitumor effect of a novel adeno-associated virus vector targeting to telomerase activity in tumor cells

The development of safe and efficient gene transfervehicles is critical for gene therapy on cancer [1]. A vastmajority of these developments have been attemptedby using adenoviral vectors and retroviral vectors. Adeno-associated virus (AAV)-based vectors are recently emer-ging nonpathogenic vectors with potential for cancergene therapy. AAV belongs to the parvovirus family,a group of small single-stranded DNA viruses, with manyadvantageous features for human gene therapy. AAV wasdiscovered…     

Retroelement particles as purification, presentation and targeting vehicles.

The manipulation of retrotransposon and retroviral particles to carry biologically active molecules is becoming feasible. In addition, recent experiments suggest that it may be possible to target these engineered particles to specific cell types. This has implications for gene therapy, biological drug delivery and vaccine design.     

Inhibition of mesothelin as a novel strategy for targeting cancer cells

Mesothelin, a differentiation antigen present in a series of malignancies such as mesothelioma, ovarian, lung and pancreatic cancer, has been studied as a marker for diagnosis and a target for immunotherapy. We, however, were interested in evaluating the effects of direct targeting of Mesothelin on the viability of cancer cells as the first step towards developing a novel therapeutic strategy. We report here that gene specific silencing for Mesothelin by distinct methods (siRNA and microRNA) decreased viability of cancer cells from different origins such as mesothelioma (H2373), ovarian cancer (Skov3 and Ovcar-5) and pancreatic cancer (Miapaca2 and Panc-1). Additionally, the invasiveness of cancer cells was also significantly decreased upon such treatment. We then investigated pro-oncogenic signaling characteristics of cells upon mesothelin-silencing which revealed a significant decrease in phospho-ERK1 and PI3K/AKT activity. The molecular mechanism of reduced invasiveness was connected to the reduced expression of β-Catenin, an important marker of EMT (epithelial-mesenchymal transition). Ero1, a protein involved in clearing unfolded proteins and a member of the ER-Stress (endoplasmic reticulum-stress) pathway was also markedly reduced. Furthermore, Mesothelin silencing caused a significant increase in fraction of cancer cells in S-phase. In next step, treatment of ovarian cancer cells (OVca429) with a lentivirus expressing anti-mesothelin microRNA resulted in significant loss of viability, invasiveness, and morphological alterations. Therefore, we propose the inhibition of Mesothelin as a potential novel strategy for targeting human malignancies.     

In vitro assessment of praziquantel and a novel nanomaterial against protoscoleces of Echinococcus granulosus

The present study describes the activity of a nanomaterial on protoscoleces of Echinococcus granulosus, which exhibited morphological changes and apoptosis. Apoptotic changes were deduced on the basis of effector caspase activation and nucleosomal laddering. Invaginated protoscoleces maintained in vitro became evaginated and had hooks, presumptive suckers and stalks. Degenerative changes of protoscoleces were evidenced after treatment with praziquantel and nano-combination. Protoscoleces treated with praziquantel had distinct attestation of necrosis and nano-combination-treated protoscoleces had signatures of apoptosis.     

A novel peptide, THALWHT, for the targeting of human airway epithelia

Targeting gene vectors to human airway epithelial cells may help to overcome the current inefficiency of gene transfer as the major problem confronting cystic fibrosis gene therapy. To elucidate novel ligands targeting abundant, apically located receptors on airway epithelial cells, a phage display library was screened for peptides binding with high affinity to such cells. This screening yielded a selectively enriched amino acid sequence, Thr-His-Ala-Leu-Trp-His-Thr (THALWHT). Subsequent binding studies confirmed that THALWHT-displaying phages bound much stronger than phages displaying control peptides to human airway epithelial cells. In contrast, no significant binding differences were observed on a variety of non-airway-derived human cell lines suggesting selective binding of the THALWHT motif to airway epithelia. Confocal microscopy of such cells after exposure to labelled synthetic THALWHT peptide indicated that its binding is followed by specific internalisation via endocytosis. A synthetic peptide comprising a cyclic CTHALWHTC domain and a DNA binding moiety enabled efficient targeted gene delivery into human airway epithelial cells. Competition assays with free THALWHT peptide confirmed the specificity of gene delivery. Thus, the THALWHT motif may prove a useful targeting moiety for both non-viral and viral gene therapy vectors.     

Fabrication of novel combinatorial drug encapsulated micelles for enhanced tumor targeting in intestinal cancer in mouse model

Hindrance to successful therapy of colon cancer is generally characterized with reduced potency of a single drug at the active site of cancer, poor drug release, and most importantly, potential toxic side effects of the drug resulting in cytotoxicity. Therefore, we investigated combinatorial drug micelles which are a potent combination of twin anticancer drugs (indomethacin and piroxicam, IND+PIR mc) for successful therapeutics of colon cancer. The novel combinatorial micelles showed improved drug encapsulation efficiency, an in vitro burst release of the dual drugs, increased cytocompatibility and increased efficacy in tumor reduction (weight and volume) than in single drug micelles (IND mc or PIR mc). The improved IND+PIR MC were to have small size 150.36 ± 15.13 nm (to avoid being taken up by liver, lungs or kidney or to sediment) with poly dispersity index (PDI) value at 0.24 ± 0.01. The PDI values suggest homogenous distribution. Encapsulation efficiency of IND+PIR mc was calculated at 86%. IND+PR mc had improved biocompatibility as demonstrated by CRL-1459™ (normal colon) cell line than IND mc or PIR mc individually. The in vivo studies in mice model clearly depict that subcutaneous tumor weight reduced by almost 75% and volume reduced drastically by 55% on administration of IND+PIR mc than IND mc or PIR mc. Furthermore, fewer side effects were found with IND+PIR mc. To conclude, IND+PIR mc may be a potential anticancer strategy to be explored more in the future.     

Cyclic RGD functionalized gold nanoparticles for tumor targeting

Integrin α(v)β(3) is an adhesion molecule involved in physiological and pathological angiogenesis as well as tumor invasion and metastasis. Therefore, it is considered an important target for molecular imaging and delivery of therapeutics for cancer, and there is a strong interest in developing novel agents interacting with this protein. Nevertheless, the interaction of individual ligands is often still weak for efficient tumor targeting, and many research groups have synthesized multivalent displays in order to overcome this problem. Gold nanoparticles can be considered a smart platform for polyvalent presentation on account of their globular shape, tunable size, facile surface chemistry, and biocompatibility. Moreover, their unique physical properties render gold nanoparticles ideal candidates for tumor diagnosis and therapy. Here, we report the synthesis and characterization of gold nanoparticles functionalized with cRGD integrin ligand and their employment for targeting human cancer cells expressing α(v)β(3) integrin.     

Nanoconjugate based on polymalic acid for tumor targeting

A new prototype of polymer-derived drug delivery system, the nanoconjugate Polycefin, was tested for its ability to accumulate in tumors based on enhanced permeability and retention (EPR) effect and receptor mediated endocytosis. Polycefin was synthesized for targeted delivery of Morpholino antisense oligonucleotides into certain tumors. It consists of units that are covalently conjugated with poly(beta-l-malic acid) (M(w) 50,000, M(w)/M(n) 1.3) highly purified from cultures of myxomycete Physarum polycephalum. The units are active in endosomal uptake, disruption of endosomal membranes, oligonucleotide release in the cytoplasm, and protection against enzymatic degradation in the vascular system. The polymer is biodegradable, non-immunogenic and non-toxic. Polycefin was also coupled with AlexaFluor 680 C2-maleimide dye for in vivo detection. Nude mice received subcutaneous injections of MDA-MB 468 human breast cancer cells into the left posterior mid-dorsum or intracranial injections of human glioma cell line U87MG. Polycefin at concentration of 2.5mg/kg was injected via the tail vein. In vivo fluorescence tumor imaging was performed at different time points, 0-180 min up to 24h after the drug injection. The custom-made macro-illumination imaging MISTI system was used to examine the in vivo drug accumulation in animals bearing human breast and brain tumors. In breast tumors the fluorescence signal in large blood vessels and in the tumor increased rapidly until 60 min and remained in the tumor at a level 6 times higher than in non-tumor tissue (180 min) (p<0.003). In brain tumors drug accumulated selectively in 24h without any detectable signal in non-tumor areas. The results of live imaging were corroborated histologically by fluorescence microscopic examination of various organs. In addition to tumors, only kidney and liver showed some fluorescent signal.     

Macrocyclic chelator assembled RGD multimers for tumor targeting

Macrocyclic chelators have been extensively used for complexation of metal ions. A widely used chelator, DOTA, has been explored as a molecular platform to assemble multiple bioactive peptides in this paper. The multivalent DOTA-peptide bioconjugates demonstrate promising tumor targeting ability.     

A novel tag-free probe for targeting molecules interacting with a flavonoid catabolite

3,4-Dihydroxyphenylacetic acid (DOPAC) is one of the colonic microflora-produced catabolites of quercetin 4′-glucoside (Q4′G). Although the interaction of DOPAC with cellular proteins might be involved in its biological activity, the actual proteins have not yet been identified. In this study, we developed a novel tag-free DOPAC probe to label the targeted proteins by the copper(I)-catalyzed azide alkyne cycloaddition (CuAAC) and verified its efficacy. Various labeled proteins were detected by the DOPAC probe with the azide labeled biotin and a horseradish peroxidase (HRP)-streptavidin complex. Furthermore, a pulldown assay identified Keap1 and aryl hydrocarbon receptor (AhR) as the target proteins for the phase 2 enzyme up-regulation.     

Design,synthesis, and in vitro evaluation of a binary targeting MRI contrast agent for imaging tumor cells

A binary targeting vector that consists of peptide sequences of Arg-Gly-Asp (RGD) and Asn-Gly-Arg (NGR) motifs has been designed and synthesized using solid-phase peptide synthesis procedure. The vector is then coupled with Gd-DOTA to work as a targeting contrast agent (CA1) for magnetic resonance imaging of human lung adenocarcinoma cells A549. Its longitudinal relaxivity is measured to be 7.55 mM^?1 s^?1 in aqueous solution at a magnetic field of 11.7 T, which is higher than that of Magnevist (4.25 mM^?1 s^?1) in the same conditions. The cell experiment shows, at the same concentration, uptake quantity of CA1 by A549 is much more than Magnevist and also superior over CA2 (a single targeting contrast agent contains only RGD). The uptake can be blocked by the targetable peptide containing RGD or NGR without coupling Gd. To summarize, CA1 has very good ability to target A549 and higher relaxivity than that of Magnevist. So CA1 is promising MRI contrast agent for high-resolution MR molecular imaging of human lung adenocarcinoma A549 cells.