KB3-1 Cells Research Articles

Targeting lysosomes by design: novel N-acridine thiosemicarbazones that enable direct detection of intracellular drug localization and overcome P-glycoprotein (Pgp)-mediated resistance.

Innovative N-acridine thiosemicarbazones (NATs) were designed along with their iron(iii), copper(ii), and zinc(ii) complexes. Lysosomal targeting was promoted by specifically incorporating the lysosomotropic Pgp substrate, acridine, into the thiosemicarbazone scaffold to maintain the tridentate N, N, S-donor system. The acridine moiety enables a significant advance in thiosemicarbazone design, since: (1) it enables tracking of the drugs by confocal microscopy using its inherent fluorescence; (2) it is lysosomotropic enabling lysosomal targeting; and (3) as acridine is a P-glycoprotein (Pgp) substrate, it facilitates lysosomal targeting, resulting in the drug overcoming Pgp-mediated resistance. These new N-acridine analogues are novel, and this is the first time that acridine has been specifically added to the thiosemicarbazone framework to achieve the three important properties above. These new agents displayed markedly greater anti-proliferative activity against resistant Pgp-expressing cells than very low Pgp-expressing cells. The anti-proliferative activity of NATs against multiple Pgp-positive cancer cell-types (colon, lung, and cervical carcinoma) was abrogated by the third generation Pgp inhibitor, Elacridar, and also Pgp siRNA that down-regulated Pgp. Confocal microscopy demonstrated that low Pgp in KB31 (-Pgp) cells resulted in acridine's proclivity for DNA intercalation promoting NAT nuclear-targeting. In contrast, high Pgp in KBV1 (+Pgp) cells led to NAT lysosomal sequestration, preventing its nuclear localisation. High Pgp expression in KBV1 (+Pgp) cells resulted in co-localization of NATs with the lysosomal marker, LysoTracker™, that was significantly (p < 0.001) greater than the positive control, the di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC) Zn(ii) complex, [Zn(DpC)2]. Incorporation of acridine into the thiosemicarbazone scaffold led to Pgp-mediated transport into lysosomes to overcome Pgp-resistance.

Bioactive, degradable and multi-functional three-dimensional membranous scaffolds of bioglass and alginate composites for tissue regenerative applications.

With a worldwide increase in the aged populace and associated geriatric diseases, there is an enormous need for the regeneration of degenerated organ systems. For this purpose, bioactive glass particulate (nBG) integrated alginate (Alg) composite membrane scaffolds were fabricated by a sol-gel assisted freeze-drying method and validated for their multifunctional utility in regenerative medicine. The presence of the combeite highly crystalline structure of nBG and Alg amorphous broad peaks were confirmed. Repetitive peaks from acids along with stretching confirmed the chemical interactions of the composites. Swelling ability, porosity, and in vitro degradation and biomineralization were analysed for up to 7 days. The results indicated that reduced swelling and degradation enhanced apatite formation. Hemocompatibility and the hemostatic properties on scaffolds were also systematically investigated. Additionally, significant cyto-compatibility and proliferation were noted in a culture with KB3-1. Further 3-D co-cultures with HDF cells and KB3-1 cells exhibited spheroid formation on Alg, nBG/Alg and nBG-Zr/Alg with profound dynamism required to establish organoids of interest. Thus, the results indicate that these 3D hydrogel membranes could offer infinite possibilities in the field of regenerative medicine, notably as an extracellular matrix (ECM) supporting the regeneration of bone, intra-vascularization, and neo-tissue formation, such as cartilage and ligaments.

Anti-proliferative Activities of Some Bivalent Symmetrical 5-Substituted Hydantoin Derivatives towards Human Brain Glioma U251 Cells (U251) and Human Carcinoma Cells (KB3-1).

Novel bivalent twin-drug type hydantoin derivatives were evaluated in vitro using a human brain glioma cell line (U251) and a human carcinoma cell line (KB3-1). Among the 5-substituted hydantoin derivatives (1a-b and 2a-d) examined in this study, bivalent symmetrical 5-substituted hydantoin derivative 1b showed the highest anti-proliferative activity towards both U251 and KB3-1 cells. The values of anti-proliferative activity (IC50) of this hydantoin derivative against the two cell lines (U251 and KB3-1) were 0.46 and 5.21 µM, respectively. The anti-proliferative activity of all of the compounds except for compounds 2a and 2d against U251 cells was higher than that of cisplatin. Bivalent symmetrical compound 1b had a biphenylmethane linker in the molecule. All of the tested bivalent hydantoin derivatives showed higher activity against U251 cells than against KB3-1 cells. For twin-drug type hydantoin derivatives 2a-d, which have a linear methylene linker in the molecules, it was found that methylene linker length in these molecules have an effect on the anti-proliferative activity against U251 and KB3-1 cells.

Anti-proliferative Activities towards Human Brain Glioma U251 Cells and Human Carcinoma Cells (KB3-1) of Some Twin-Drug Type Bivalent C2-Symmetrical Phenylboronic Acid Derivatives.

Derivatives of C2-symmetrical bivalent phenylboronic acid exhibit several remarkable biological activities such as anti-herpes simplex virus (HSV)-1 and cytotoxic activities against Vero cells and they can reverse the effect of anticancer drugs. Novel symmetrical bivalent molecules were synthesized and their biological activities were evaluated in vitro using a human brain glioma cell line (U251) and a human carcinoma cell line (KB3-1). Among the tested compounds (1a-i), bivalent C2-symmetrical phenylboronic acid derivative 1g showed the highest anti-proliferative activity towards both U251 and KB3-1 cells. The values of 50% anti-proliferative activity (IC50) of this compound against the two cell lines (U251 and KB3-1) were 19.0 and 3.78 µM, respectively. The anti-proliferative activity of compound 1g towards KB3-1 cells was higher than that of cisplatin. The bivalent C2-symmetrical compound 1g had a linear methylene linker in the molecule.

Radiosynthesis and evaluation of a 99mTc-folic acid radiotracer prepared using [99mTcN(PNP)]2+ metal fragment

Folate receptors (FR) are over-expressed on a wide variety of tumor cells and are a potential molecular target for radiolabeled folates. In this respect, several SPECT and PET based radiofolates have been evaluated in the past albeit with their high renal uptake posing limitation towards their clinical use. To overcome this, a new 99mTc labeled folic acid was synthesized via the use of [99mTcN(PNP)]2+ metal fragment, where the presence of the latter pharmacophore redirects in vivo clearance via the hepatobiliary pathway. In this respect, folic acid was derivatized at the γ-acid group with a cysteine BFCA (bifunctional chelating agent) and subsequently reacted with the preformed [99mTcN]2+ intermediate in presence of PNP2 (bisphosphine) ligand, to yield the final complex. While preliminary, in vivo distribution of the complex exhibited high association of activity with liver and intestines and provided support to the rationality of the present design as clearance of labeled folic acid could be effected via the hepatic route, the in vitro studies of the folic acid-cysteine conjugate carried out in KB-31 cells, did not show much promise with reduction in receptor affinity in comparison with the native folic acid. The route followed herein to prepare a folic-acid based radiotracer constitutes the first report of radiolabeling folic acid using the [99mTcN(PNP)]2+ as a radiosynthon. Modification in the structure of conjugate by linking the BFCA through a long-chain linker can be envisaged to improve the affinity of [99mTcN(PNP)]-folic acid complex towards FRs.

Abstract 4750: Overcoming DDP resistance using folic acid-heparin-DDP nanoparticles

Abstract Cisplatin (DDP) has been widely used in the treatment of different cancers. However, resistance to DDP limits its clinical application. Since reduced intracellular DDP accumulation is one of the main causes for DDP resistance, increasing the accumulation of DDP may overcome drug resistance. The folate receptor (FR) is overexpressed on the surface of many human tumors including breast, ovarian and lung cancer, but is found at low levels in normal tissues. In this study, folic acid (FA) was conjugated to heparin-DDP (FHDDP) nanoparticles to target FR. In vitro cellular uptake experiments showed that the accumulation of FHDDP nanoparticles in FR-expressing DDP-resistant KB-CP.5 cancer cells (selected from KB3-1 cells, and showing a 36-fold increase in resistance to DDP compared with KB3-1) was significantly higher than that of free DDP (9.31±2.2 ng Pt/106 cells vs 0.98±0.25 ng Pt/106 cells, Pt:0.5ug/ml, 4 hrs at 37°C). Further, the level of platinum DNA-adducts in the resistant cells treated with FHDDP nanoparticles was about 4-fold greater than in cells treated with free DDP (24.21±5.32 pg Pt/ug DNA vs 97.9±22.5 pg Pt/ug DNA). Nude mice bearing KB-CP.5 cell tumors receiving FHDDP nanoparticles (2.5mg/kg, tail vein injection, twice weekly, 5 times) showed enhanced antitumor effects as compared with mice treated with free DDP (tumor volume: 823±221mm3 vs 1450±392mm3, 21 days after treatment), while non-targeted heparin-DDP nanoparticles did not show significant difference compared with free DDP. Our results suggest that FHDDP nanoparticles have the potential to be used for overcoming DDP resistance.(This research was supported by the National Cancer Institute award 5 P50 CA128613) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4750. doi:1538-7445.AM2012-4750

Use of Size and a Copolymer Design Feature To Improve the Biodistribution and the Enhanced Permeability and Retention Effect of Doxorubicin-Loaded Mesoporous Silica Nanoparticles in a Murine Xenograft Tumor Model

A key challenge for improving the efficacy of passive drug delivery to tumor sites by a nanocarrier is to limit reticuloendothelial system uptake and to maximize the enhanced permeability and retention effect. We demonstrate that size reduction and surface functionalization of mesoporous silica nanoparticles (MSNP) with a polyethyleneimine-polyethylene glycol copolymer reduces particle opsonization while enhancing the passive delivery of monodispersed, 50 nm doxorubicin-laden MSNP to a human squamous carcinoma xenograft in nude mice after intravenous injection. Using near-infrared fluorescence imaging and elemental Si analysis, we demonstrate passive accumulation of ∼12% of the tail vein-injected particle load at the tumor site, where there is effective cellular uptake and the delivery of doxorubicin to KB-31 cells. This was accompanied by the induction of apoptosis and an enhanced rate of tumor shrinking compared to free doxorubicin. The improved drug delivery was accompanied by a significant reduction in systemic side effects such as animal weight loss as well as reduced liver and renal injury. These results demonstrate that it is possible to achieve effective passive tumor targeting by MSNP size reduction as well as by introducing steric hindrance and electrostatic repulsion through coating with a copolymer. Further endowment of this multifunctional drug delivery platform with targeting ligands and nanovalves may further enhance cell-specific targeting and on-demand release.

Autonomous in vitro anticancer drug release from mesoporous silica nanoparticles by pH-sensitive nanovalves.

Mesoporous silica nanoparticles (MSNP) have proven to be an extremely effective solid support for controlled drug delivery on account of the fact that their surfaces can be easily functionalized in order to control the nanopore openings. We have described recently a series of mechanized silica nanoparticles, which, under abiotic conditions, are capable of delivering cargo molecules employing a series of nanovalves. The key question for these systems has now become whether they can be adapted for biological use through controlled nanovalve opening in cells. Herein, we report a novel MSNP delivery system capable of drug delivery based on the function of beta-cyclodextrin (beta-CD) nanovalves that are responsive to the endosomal acidification conditions in human differentiated myeloid (THP-1) and squamous carcinoma (KB-31) cell lines. Furthermore, we demonstrate how to optimize the surface functionalization of the MSNP so as to provide a platform for the effective and rapid doxorubicin release to the nuclei of KB-31 cells.

Novel Nanoassemblies Composed of Squalenoyl−Paclitaxel Derivatives: Synthesis, Characterization, and Biological Evaluation

Using the anticancer compound paclitaxel as a model drug, this study investigates the potential of the squalenoylation technology (i.e., bioconjugation with the natural lipid squalene) in addressing the drug ability and delivery issues of poorly soluble therapeutic agents. In this view, a variety of novel squalene-based prodrugs of the anticancer compound paclitaxel were synthesized, which produced nanoparticles in water. These prodrugs were obtained by covalent coupling of the paclitaxel 2'-hydroxyl group as direct ester, as well as with a succinate or a diglycolate ester as cleavable linker to the 1,1',2-tris-norsqualenoic acid. The hydrophilicity of these paclitaxel bioconjugates was increased by placing poly(ethylene glycol) chains of different lengths between paclitaxel and the squalenoyl moiety. All these prodrugs self-assembled into nanosized aggregates in aqueous solution as characterized by dynamic light scattering, atomic force microscopy, and transmission electron microscopy. The critical aggregation concentration was very low, ranging from 0.09 to 0.4 mg/L. Zeta potential measurements revealed that all squalenoyl-paclitaxel nanoassemblies (NA) held a global negative charge and appeared stable in water for several weeks as determined by particle size measurement. The release of paclitaxel from NA was evaluated in different conditions and in the presence of serum and depended on the nature of the linker used. Preliminary biological assessment showed that these squalenoyl-paclitaxel NA induced the formation of microtubule bundles in HT-29 and KB-31 cells, and additionally displayed notable cytotoxicity on a lung tumor cell line. Furthermore, the cytotoxic activity of these different prodrugs correlated closely with the observed linker stability. Overall, the squalenoylation nanotechnology opens up interesting perspectives for the development of injectable prodrugs of poorly soluble therapeutic compounds by addressing the associated physicochemical and biopharmaceutical challenges.

A Convenient Thiazole Orange Fluorescence Assay for the Evaluation of DNA Duplex Hybridization Stability

A simple and rapid method for measuring the hybridization stability of duplexes of DNAs and other oligomers in different environments is described. When added to an oligomer duplex, the thiazole orange (TO) dye intercalates and in this state is fluorescent. Therefore, when duplex dissociation occurs, the release of TO results in a detectable change in fluorescence intensity. This assay was developed primarily to screen antisense oligomer duplexes that are stable in serum and in the cytoplasm but unstable in the presence of their target messenger RNA (mRNA). The two antisense oligomers of this investigation were both 25 mer phosphorothioate (PS) DNAs, one directed against the RIalpha mRNA and the other directed against the mdr1 mRNA. The former duplex was first used in the solution studies, in most cases duplexed with a 16 mer phosphodiester (PO) complementary DNA (i.e., PS-DNA25/PO-cDNA16). Both duplexes were then tested in a series of cell studies using SK-BR-3 (RIalpha+), KB-G2 (mdr1++), and KB-31 (mdr1+/-) cells. Preliminary measurements in solution showed that maximum fluorescence was achieved when more than ten TO molecules were bound to each duplex. When a 25 mer PO-DNA or PO-RNA with the base sequence of the RIalpha mRNA was added, the dramatic change in fluorescence intensity that followed signified dissociation of the antisense DNA from the study duplex and reassociation with the target DNA. Kinetic measurements showed that this process was completed in about 3 min. Fluorescent measurements of SK-BR-3 (RIalpha+) cells incubated at 37 degrees C with the anti-RIalpha study duplex over time showed a maximum at the point where the loss of fluorescence due to dissociation of the study duplex, probably by an antisense mechanism, began to dominate over the increasing fluorescence due to continuing cellular accumulation. A similar result was observed in the KB-G2 (mdr1+) cells incubated with the anti-mdr1 study duplex. When study duplexes shown to be stable in serum were incubated with their target cells, the assay successfully detected evidence of dissociation, most likely by an antisense mechanism. Thus, a TO fluorescence assay has been developed that is capable of detecting the dissociation of DNA duplexes.

Cytoplasmic and Nuclear Accumulation of Antisense Oligomers Incubated as Streptavidin Delivery Nanoparticles

The cytoplasmic accumulation and nuclear targeting of antisense oligomers were evaluated in cell culture following incubation with the cell penetrating peptide tat or polyargenine as a two-component delivery nanoparticle using streptavidin as linker. The fluorescent labeled uniform phosphodiester (PO), phosphorothioate (PS) and MORF oligomers were antisense to the mdr1 mRNA and were studied in KB-G2 (Pgp++) and KB-31 (Pgp+/-) cells. Fluorescence microscopy showed much higher cellular accumulation using the antisense compared to the sense PS/tat nanoparticle. The distribution in cells receiving either the PS or MORF, but not the PO, antisense oligomers in all of their forms showed definite migration to the nucleus. Incubations in KB-G2 cells of all three antisense oligomers with streptavidin as the onecomponent or including tat as the two-component nanoparticles led to increases in Rhodamine 123 accumulation by 200∼250% compared to free oligomers, almost certainly due to antisense interference with Pgp function. Thus presence of the streptavidin linker had no obvious detrimental influence on the functions of the carriers or the antisense oligomers. Since nuclear migration appears to be a characteristic property of oligomers, delivery via the nanoparticle may be a useful method of increasing the accumulation into the nucleus of antisense oligomers radiolabeled for Auger emittingbased radiotherapy.

Pyripyropenes, Fungal Sesquiterpenes Conjugated with .ALPHA.-Pyrone and Pyridine Moieties, Exhibits Anti-angiogenic Activity against Human Umbilical Vein Endothelial Cells

In the course of our search for anti-angiogenic substances, pyripyropenes A (1), B (2), and D (3) were re-discovered as selective anti-proliferative substances against human umbilical vein endothelial cells (HUVECs) from a marine-derived fungus of Aspergillus sp. Pyripyropenes showed potent anti-proliferative activity against HUVECs with IC(50) values of the range of 0.1-1.8 muM, which were cytostatic at 0.05 to 20 muM. The selective index was more than 55-fold in comparison with those of several tumor cell lines. Compound 1 inhibited vascular endothelial growth factor (VEGF)-induced migration and tubular formation of HUVECs, while 1 showed no effect on the VEGF-induced phosphorylations of extracellular signal-regulated kinase (ERK)1/2, p38, and Akt. Pyripyropenes were originally isolated as an inhibitor of acyl-CoA: cholesterol acyltransferase (ACAT-2). While, the expression level of ACATs between HUVECs and other tumor cell lines did not correspond to the selective index of the anti-proliferative activity of compound 1. Moreover, ACATs inhibitor, 2,2-dimethyl-N-(2,4,6-trimethoxyphenyl)dodecanamide (CI-976), showed growth inhibitory activity with only poor selectivity (2.4-fold) between HUVECs and human epidermoid carcinoma KB3-1 cells.

IK1 channel activity contributes to cisplatin sensitivity of human epidermoid cancer cells

Cisplatin, a platinum-based drug, is an important weapon against many types of cancer. It induces apoptosis by forming adducts with DNA, although many aspects of its mechanism of action remain to be clarified. Previously, we found a role for the volume-sensitive, outwardly rectifying Cl(-) channel in cisplatin-induced apoptosis. To investigate the possibility that cation channels also have a role in the cellular response to cisplatin, we examined the activity of cation channels in cisplatin-sensitive KB-3-1 (KB) epidermoid cancer cells by the whole cell patch-clamp method. A cation channel in KB cells, activated by hypotonic stress, was identified as the Ca2+-activated, intermediate-conductance K+ (IK1) channel on the basis of its requirement for intracellular Ca2+, its blockage by the blockers clotrimazole and triarylmethane-34, and its suppression by a dominant-negative construct. Activity of this channel was not observed in KCP-4 cells, a cisplatin-resistant cell line derived from KB cells, and its molecular expression, observed by semiquantitative RT-PCR and immunostaining, appeared much reduced. Cell volume measurements confirmed a physiological role for the IK1 channel as a component of the volume-regulatory machinery in KB cells. A possible role of the IK1 channel in cisplatin-induced apoptosis was investigated. It was found that clotrimazole and triarylmethane-34 inhibited a cisplatin-induced decrease in cell viability and increase in caspase-3/7 activity, whereas 1-ethyl-2-benzimidazolinone, an activator of the channel, had the opposite effect. Thus IK1 channel activity appears to mediate, at least in part, the response of KB cells to cisplatin treatment.

Cell Culture and Xenograft-Bearing Animal Studies of Radiolabeled Antisense DNA Carrier Nanoparticles with Streptavidin as a Linker

Transmembrane transfectors (carriers) are increasingly being viewed as helpful or even necessary to improve cellular delivery in connection with antisense tumor targeting and other applications requiring cell membrane transport of DNAs, RNAs, and other oligomers. We are investigating streptavidin as a convenient linker for biotinylated carriers and oligomers because it requires only simple mixing for preparation. The goal of this study was to evaluate antisense DNA-streptavidin-carrier nanoparticles for accumulation in cell culture and in xenograft-bearing mice. The 3 carriers were cholesterol, a 10-mer Tat peptide, and a 10-mer polyarginine peptide. A 20-mer DNA targeting the mdr1 messenger RNA coding for Pgp expression was used as the phosphodiester (PO) DNA as well as the phosphorothioate (PS) DNA. In all cases, the (99m)Tc radiolabel was on the DNA. The 8 nanoparticles were first tested in mdr1(++) KB-G2 and TCO-1 cells and in mdr1(+/-) KB-31 cells in culture for evidence of improved accumulation and antisense targeting. Thereafter, the PS DNA-streptavidin-Tat, PO DNA-streptavidin-Tat, and PS DNA-streptavidin-cholesterol nanoparticles were administered intravenously to KB-G2 xenograft-bearing mice, and tissue distributions were measured. In culture, the PO nanoparticles showed increased accumulation compared with the corresponding nanoparticles without the carrier in all 3 cell types; in contrast, with the PS nanoparticles, any similar carrier-mediated increase may have been obscured by the much higher protein-binding affinity of PS DNA. As evidence of antisense targeting, the Tat and cholesterol PS nanoparticles showed statistically significant accumulation at 23 h in cells in the descending order TCO-1, KB-G2, and KB-31, although there were no significant differences among the PO nanoparticles. In xenograft-bearing mice, the tissue accumulation of both forms of the PS nanoparticles greatly exceeded that of the PO nanoparticles and, including in the tumor, were similar to that obtained previously for naked PS DNA. The presence of the streptavidin linker had no obvious detrimental effect on the functions of the carriers and antisense DNAs. The higher protein-binding affinity of the PS nanoparticles than the PO nanoparticles was still apparent both in vitro and in vivo, the pharmacokinetics of the PS nanoparticles were similar to that of naked PS DNA, and the carriers improved cellular accumulation, at least for the PO nanoparticles. These observations, taken together with the higher accumulation of both forms of the antisense PS nanoparticles in mdr1(++) KB-G2 and TCO-1 cells than in mdr1(+/-) KB-31 cells, suggest that further effort is justified to confirm that the antisense properties of the DNAs were not compromised by the presence of streptavidin.

Optical Antisense Imaging of Tumor with Fluorescent DNA Duplexes

Antisense targeting of tumor with fluorescent conjugated DNA oligomers has the potential of improving tumor/normal tissue ratios over that achievable by nuclear antisense imaging. When administered as a linear duplex of two fluorophore-conjugated oligomers arranged in a manner that inhibits fluorescence as the duplex and designed to dissociate only in the presence of the target mRNA, the fluorescence signal should in principle be inhibited everywhere except in the target cell. Optical imaging by fluorescence quenching using linear fluorophore-conjugated oligomers has not been extensively investigated and may not have been previously considered for antisense targeting. We evaluated in cell culture and in KB-G2 tumor bearing nude mice a 25-mer phosphorothioate (PS) anti- mdr1 antisense DNA conjugated with the Cy5.5 emitter on its 3' equivalent end and hybridized as a linear duplex with a shorter 18-mer phosphodiester (PO) complementary DNA (cDNA) with the Black Hole inhibitor BHQ3 on its 5' end. In serum environments, 90% of the DNA25-Cy5.5 fluorescence was inhibited immediately following addition of the cDNA18-BHQ3 and showed only slight loss of inhibition over 24 h at 37 degrees C. As evidence of antisense specific binding, when incubated with the DNA25-Cy5.5/cDNA18-BHQ3 duplex, the fluorescence was lower in KB-31 (Pgp +/-) cells compared to KB-G2 (Pgp++) cells, but when incubated with the control cDNA18-Cy5.5/DNA25-BHQ3 duplex in which the fluorophores were reversed, the fluorescence of both cell types was low. As further evidence of specific binding, the fluorescent intensity of total RNA from KB-G2 cells incubated with the study duplex showed evidence of dissociation and hybridization with the target mRNA. Furthermore, the fluorescence microscopy images of KB-G2 cells incubated with DNA25-Cy5.5 as the singlet or study duplex show that migration in both cases is to the nucleus. The animal studies were performed in mice bearing KB-G2 tumor in one thigh and receiving iv the study or control duplexes. The tumor/normal thigh fluorescence ratio was clearly positive as early as 30 min postinjection in the study mice and reached a maximum at 5 h. By contrast, much lower fluorescence was observed in mice receiving the control duplex at the same dosage. Fluorescence microscope imaging showed that the Cy5.5 fluorescence was much higher in tumor sections from the animal that had received the study rather than control duplex. Thus combining a fluorophore-conjugated antisense DNA with an inhibitor-conjugated shorter complementary cDNA inhibited fluorescence both in cell culture and in tumored animals except in the presence of the target mRNA. This proof of concept investigation of optical antisense targeting therefore suggests that further studies including optimization of this approach are appropriate.

Cationic Transfectors Increase Accumulation in Cultured Tumor Cells of Radiolabeled Antisense DNAs Without Entrapment

While cellular accumulations in culture of oligomers, such as interfering RNA and antisense DNA, are reported to benefit from the addition of transmembrane transfectors (TFs), the extent to which individual TFs improve cellular delivery is usually inferred and rarely measured. The goal of this investigation was to use radioactivity to measure in cells in culture the degree to which accumulations of DNA increased when complexed with TFs and without DNA entrapment in vesicles. The antisense (AS) DNA targeting mdr1 mRNA coding for P-glycoprotein (Pgp) and its sense (S) complement DNA were radiolabeled with 99mTc and mixed with jetPEI, Chariot, or Neophectin over a range of TF/DNA ratios. Thereafter,the radiolabeled DNAs with and without TFs were incubated with KB-G2 (mdr1(+/+)) and KB-31 (mdr(+/-))cells at 37 degrees C in serum or serum-free media for 20-24 hours at a fixed DNA concentration of 13 nM. Cellular accumulations were increased under most incubation conditions and by as much as threefold with jetPEI and eightfold with Neophectin. As evidence against entrapment, the accumulations of AS DNAs were higher than S DNAs in virtually all measurements and higher in virtually all accumulations in the mdr1(+/+) cells, compared to the mdr1(+/-) cells. In conclusion, by using radiolabeled DNAs, definitive evidence was obtained showing that the addition of Neophectin and jet PEI increased cellular accumulations of both AS and S DNA without evidence of vesicle entrapment.

Biological characterization of folic acid-conjugated poly(H2NPEGCA-co-HDCA) nanoparticles in cellular models

The folate receptor (FR) is a highly selective tumor marker over expressed in many human cancers and it constitutes a useful target for tumor-specific drug delivery. Thus, the conjugation of folic acid to different drugs or drug carriers may enhance the delivery of the therapeutic agent to FR-positive tumor cells. The aim of this study was to investigate the interactions of folate-conjugated polyalkylcyanoacrylate nanoparticles with tumor cells overexpressing the FR. For this purpose, nanoparticles were prepared by nanoprecipitation of the poly[aminopoly(ethylene glycol) cyanoacrylate-co-hexadecyl cyanoacrylate] [poly(H2NPEGCA-co-HDCA)] copolymer and labeled with the hydrophobic fluorescent dye nile red. Nile red-loaded nanoparticles were then conjugated to folic acid via the PEG terminal amino groups. Four human cancer cell lines were then tested by western blot in order to evaluate the FR expression levels. KB3-1 cell line showed the higher expression level, while MCF-7 cells were taken as a control. After measuring the cytotoxicity of the nanoparticles on these two cell lines, fluorescent folate-nanoparticles were incubated with them and the cellular uptake was evaluated by confocal microscopy and flow cytometry. KB3-1 cells showed a greater nanoparticle internalization, when compared to MCF-7 cells.

P53 negatively regulates the expression of FAT10, a gene upregulated in various cancers

FAT10 is a member of the ubiquitin-like modifier family of proteins and has been implicated to play important roles in antigen presentation, cytokine response, apoptosis and mitosis. We have recently demonstrated the upregulation of FAT10 gene expression in 90% of hepatocellular carcinoma patients. Here, we identified and characterized the promoter of the FAT10 gene to elucidate the mechanism of FAT10 gene expression. Notably, we found that the 5' untranslated region (5'UTR), from the transcription start site to 15 bases before the translational start site, displays significant promoter activity. Regions upstream of the 5'UTR (from +26 to -1997) do not confer any promoter activity. Curiously, FAT10 promoter activity and expression is significantly repressed in KB3-1 and HepG2 cells, which have wild-type p53, than in p53-negative Hep3B cells. The role of p53 in regulating FAT10 expression was evident by the significant downregulation (P<0.05) of FAT10 mRNA expression and promoter activity when wild-type p53 was transfected into p53-null Hep3B cells. Conversely, inhibiting p53 expression through siRNA against p53 significantly enhanced FAT10 expression and promoter activity. p53 was found to bind in vivo to the 5' half consensus sequence of p53-binding site located at the FAT10 promoter. Hence, we propose that FAT10 is a downstream target of p53 and dysregulation of FAT10 expression in p53-defective cells could contribute to carcinogenesis.

Antiplasmodial activity of ferrocenyl chalcones: Investigations into the role of ferrocene

A series of ferrocenyl chalcones were synthesized and evaluated in vitro against Plasmodium falciparum (K1) in a [ 3H] hypoxanthine uptake assay. Appropriate size, electronic, lipophilic and electrochemical parameters were determined for QSAR analysis. The results showed that the location of ferrocene influenced the ease of oxidation of Fe 2+ in ferrocene and the polarity of the carbonyl linkage. These parameters were found to influence antiplasmodial activity. A general trend was noted in which compounds with ferrocene adjacent to the carbonyl linkage (series A) were associated with more selective and potent antiplasmodial activities. These compounds had polarized carbonyl linkages, lower lipophilicities and ferrocene rings that were less readily oxidized. The most active analogue was 1-ferrocenyl-3-(4-nitrophenyl)prop-2-en-1-one ( 28) (IC 50 4.6 μM, selectivity index 37 against KB3-1 cells). To understand how the redox properties of ferrocene might influence antiplasmodial activity, the oxidant properties of selected compounds were investigated in antioxidant (ABTS +) and EPR experiments. The incorporation of ferrocene in the chalcone template was found to enhance its role in processes that involved the quenching and generation of free radicals. Thus, ferrocene may participate in redox cycling and this process may contribute to the antiplasmodial activity of ferrocenyl chalcones. However, the extent to which this property is manifested is also influenced by other physicochemical properties (lipophilicity, polarity, and planarity) of the compound.

Esters and amides of 2,3-dimethoxy-8,9-methylenedioxy-benzo[ i]phenanthridine-12-carboxylic acid: Potent cytotoxic and topoisomerase I-targeting agents

The exceptional topoisomerase I-targeting activity and antitumor activity of 5-(2- N, N-dimethylamino)ethyl-8,9-dimethoxy-2,3-methylenedioxy-5 H-dibenzo[ c, h][1,6]naphthyridin-6-one (ARC-111, topovale) prompted studies on similarly substituted benzo[ i]phenanthridine-12-carboxylic ester and amide derivatives. Among the benzo[ i]phenanthridine-12-carboxylic esters evaluated, the 2-( N, N-dimethylamino)ethyl, 2-( N, N-dimethylamino)-1-methylethyl, and 2-( N, N-dimethylamino)-1,1-dimethylethyl esters possessed similar cytotoxicity, ranging from 30 to 55 nM in RPMI8402 and KB3-1 cells. Several of the carboxamide derivatives possess potent topoisomerase I-targeting activity and cytotoxicity. The 2-( N, N-dimethylamino)ethyl, 2-( N, N-diethylamino)ethyl, and 2-(pyrrolidin-1-yl)ethyl amides were among the more cytotoxic benzo[ i]phenanthridine-12-carboxylic derivatives, with IC 50 values ranging from 0.4 to 5.0 nM in RPMI8402 and KB3-1 cells.