BCL-2 antisense and cisplatin combination treatment of MCF-7 breast cancer cells with or without functional p53

Summary Chemotherapy has been used for treatment of breast cancer but with limited success. We characterized the effects of bcl-2 antisense and cisplatin combination therapy in two human isogenic breast carcinoma cells p53(+)MCF-7 and p53(−)MCF-7/E6. The transferrin-facilitated lipofection strategy we have developed yielded same transfection efficiency in both cells. Bcl-2 antisense delivered with this strategy significantly induced more cell death, apoptosis, and cytochrome c release in MCF-7/E6 than in MCF-7, but did not affect Fas level in both cells and activated caspase-8 equally. Cisplatin exerted same effects on cell viability and apoptosis in both cells, but released smaller amounts of cytochrome c while activated more caspase-8 in MCF-7/E6. The combination treatment yielded greater effects on cell viability, apoptosis, cytochrome c release, and caspase-8 activation than individual treatments in both cells although p53(−) cells were more sensitive. The potentiated activation of caspase-8 in the combination treatment suggested that caspase-8-mediated (but cytochrome c-independent) apoptotic pathway is the major contributor of the enhanced cell killing. Thus, bcl-2 antisense delivered with transferrin-facilitated lipofection can achieve the efficacy of killing breast cancer cells and sensitizing them to chemotherapy. Bcl-2 antisense and cisplatin combination treatment is a potentially useful therapeutic strategy for breast cancer irrespective of p53 status. Hesham Basma and Hesham El-Refaey contributed equally     

Topical delivery of retinyl ascorbate: 4. Comparative anti-oxidant activity towards DPPH

The free radical scavenging properties of retinyl ascorbate (RA-AsA) were determined by monitoring the decomposition of 2,2-diphenyl-1-picrylhydrazyl (DPPH) as a function of time and in comparison with ascorbic acid (AsA), ascorbic acid palmitate (AsA-Pal), retinoic acid (RA), retinol (ROL) and retinol palmitate (Rol-Pal). The rate constant of RA-AsA (mean₃±SD) was 4.9±0.3 M^-1 s^-1, and indicated greater potency as an antioxidant compared to the rest of the test compounds (AsA 3.4±0.4 M^-1 s^-1, AsA-Pal, 2.9±0.2 M^-1 s^-1, RA 1.4±0.3 M^-1 s^-1, ROL 1.3±0.1 M^-1 s^-1, Rol-Pal exhibited insignificant activity). The decomposition rate constant of DPPH, 5±0.6 × 10^-8 M^-1 s^-1, in ethanol and BHA, 154±3 M^-1 s^-1 were both used as control. The compound RA-2-carboxy-2-hydroxy-ethanoate was isolated by prep-TLC and was identified, by ¹³C and ¹HNMR spectroscopy, as the major by-product from the reaction of RA-AsA with DPPH, which was also found to be potent antioxidant, 2.1±0.2 M^-1 s^-1. This suggests that oxidation of AsA moiety did not lead to the production of erythrulose species, which could cause deleterious modifications of cellular proteins.     

Enhanced plasmid DNA delivery using anionic LPDII by listeriolysin O incorporation

BACKGROUND: A major obstacle to achieving effective DNA-based therapeutics is efficient delivery of the DNA to its site of action in the cell. Upon internalization by endocytosis, the endosomal membrane represents a critical physical barrier preventing access of DNA to the cell cytosol. In order to overcome the membrane barrier and facilitate cytosolic entry, the endosomolytic bacterial protein listeriolysin O (LLO) is a potentially promising agent. METHODS: LLO was incorporated in an anionic liposome-entrapped polycation-condensed DNA delivery system (LPDII). Plasmid DNA was condensed using protamine sulfate and then complexed to anionic liposomes. LLO was incorporated into the delivery vehicle through encapsulation in anionic, pH-sensitive liposomes. Transfection levels were monitored using a model reporter plasmid encoding luciferase in P388D1 cells, a macrophage-like cell line. RESULTS: Transfection using the anionic LPDII delivery platform was enhanced through incorporation of LLO. Additionally, the net charge of the condensate, the lipid composition, and the total amount of LLO-liposomes were all capable of modulating the transfection levels of the vehicle. Importantly, in the presence of serum, transfection levels using the LLO-containing LPDII system were comparable to established cationic lipid delivery systems. CONCLUSIONS: LLO is capable of facilitating transfection using an anionic LPDII system. This anionic delivery vehicle represents the successful combination of the LPDII system for condensation of the DNA with the unique endosomolytic properties of LLO for improved transfection using plasmid DNA.     

Th1-type immune response to a Coccidioides immitis antigen delivered by an attenuated strain of the non-invasive enteropathogen Vibrio cholerae

The antigen-2 or proline rich antigen (Ag2/PRA) from Coccidioides immitis, known to protect mice against experimental Coccidioidomycosis, was expressed in the genetically attenuated cholera vaccine candidate Vibrio cholerae 638 and its thymine auxotrophic derivative 638T. Intranasal immunization of mice with strains producing Ag2/PRA induced serum vibriocidal antibody and Ag2/PRA-specific total IgG responses in outbred Swiss Webster and inbred BALB/c mice. Analysis of IgG subclasses showed a predominance of IgG2a subclass antibodies. Lymphocytes from immunized mice stimulated with pure Ag2/PRA showed a significant proliferative response with production of interferon-gamma. Positive selection for plasmid maintenance in vivo did not enhance immune response to Ag2/PRA. These results demonstrate that genetically attenuated strains of the non-invasive pathogen V. cholerae can be used to express and deliver foreign antigens to stimulate a Th1 type of immune response.     

Enhanced osteoclastogenesis in women after natural delivery

In the pre-expulsive and expulsive phases of labor, oxytocin and several other osteoclastogenic mediators, such as prostaglandins and IL-6, are secreted in high concentrations. This study was undertaken to assess whether the peripheral blood obtained from healthy women after vaginal delivery contains a larger pool of osteoclast precursors compared with age- and gender-matched controls. Our results clearly show that the number and size of osteoclasts generated in vitro from osteoclast precursors isolated from women after delivery are significantly larger than those from controls. This finding can account for the decrease in bone mass that is often observed during the breastfeeding period and the concomitant release of high quantities of calcium in the milk. Further investigations are required to establish whether analysis of blood osteoclast precursors can be predictive of changes in bone remodeling in this setting.     

Lecithin organogels as a potential phospholipid-structured system for topical drug delivery: A review

The purpose of this review is to give an insight into the considerable potential of lecithin organogels (LOs) in the applications meant for topical drug delivery. LOs are clear, thermodynamically stable, viscoelastic, and biocompatible jelly-like phases, chiefly composed of hydrated phospholipids and appropriate organic liquid. These systems are currently of interest to the pharmaceutical scientist because of their structural and functional benefits. Several therapeutic agents have been formulated as LOs for their facilitated transport through topical route (for dermal or transdermal effect), with some very encouraging results. The improved topical drug delivery has mainly been attributed to the biphasic drug solubility, the desired drug partitioning, and the modification of skin barrier function by the organogel components. Being thermodynamically stable, LOs are prepared by spontaneous emulsification and therefore posses prolonged shelf life. The utility of this novel matrix as a topical vehicle has further increased owing to its very low skin irritancy potential. Varied aspects of LOs viz formation, composition, phase behavior, and characterization have been elaborated, including a general discussion on the developmental background. Besides a comprehensive update on the topical applications of lecithin organogels, the review also includes a detailed account on the mechanistics of organogelling. Published: October 6, 2005     

Gene delivery to embryonic stem cells

Since the establishment of embryonic stem (ES) cells and the identification of tissue-specific stem cells, researchers have made great strides in the analysis of the natural biology of such stem cells for the development of therapeutic applications. Specifically, ES cells are capable of differentiating into all of the cell types that constitute the whole body. Thus, ES cell research promises new type of treatments and possible cures for a variety of debilitating diseases and injuries. The potential medical benefits obtained from stem cell technology are compelling and stem cell research sees a bright future. Control of the growth and differentiation of stem cells is a critical tool in the fields of regenerative medicine, tissue engineering, drug discovery, and toxicity testing. Toward such a goal, we present here an overview of gene delivery in ES cells, covering the following topics: significance of gene delivery in ES cells, stable versus transient gene delivery, cytotoxicity, suspension versus adherent cells, expertise, time, cost, viral vectors for gene transduction (lentiviruses, adenoviruses, and adeno-associated viruses, chemical methods for gene delivery, and mechanical or physical gene delivery methods (electroporation, nucleofection, microinjection, and nuclear transfer).     

Calcium enhances the transfection potency of stabilized plasmid-lipid particles

Previous work from this laboratory has shown that plasmid DNA can be encapsulated in small (70-nm-diameter) stabilized plasmid-lipid particles (SPLP) that consist of a single plasmid encapsulated within a bilayer lipid vesicle. SPLP preferentially transfect tumor tissue following intravenous administration. Although the levels of transgene expression in vivo are greater for SPLP than can be achieved with naked DNA or complexes, they are lower than may be required for therapeutic benefit. In the present work we examine whether Ca2+ can enhance the transfection potency of SPLP. It is shown that Ca2+ can enhance SPLP transfection potency in bovine hamster kidney cells by 60- to 100-fold when treated in serum containing medium and an additional 60-fold when serum is absent for the initial 10 min of the transfection period. When cells are treated with SPLP in the presence of Ca2+, there is a fivefold increase in intact plasmid in the cell. It is also shown that this Ca2+ effect involves the formation of calcium phosphate precipitates; however, these precipitates are not directly associated with the SPLP plasmid DNA. The ability of calcium phosphate to facilitate delivery of other macromolecules without direct association is also demonstrated by the release of large-molecular-weight dextrans from endosomal/lysosomal compartments in the presence of calcium phosphate. Finally, it is shown that, unlike naked DNA, SPLP transfection potency in the presence of calcium phosphate is not affected by nuclease activity.     

Cyclodextrin-polyethylenimine conjugates for targeted in vitro gene delivery

Many human gene therapies will require cell-specific targeting. Though recombinant viruses are much more efficient than nonviral vectors, the latter, especially polymers, have the advantage of being targetable via conjugation of cell-specific ligands, including sugars, peptides, and antibodies, which can be covalently attached to the polymer using a variety of chemistries. Cyclodextrin, which forms inclusion complexes with small hydrophobic molecules, has been incorporated into a gene-delivery polymer and may provide a facile and versatile attachment site for targeting ligands. Polyethylenimine (PEI) was derivatized with beta-cyclodextrin on approximately 10% of the polymer's amines (termed CD-PEI). Human insulin was also derivatized with a hydrophobic palmitate group (pal-HI), which could anchor the protein to CD-PEI/DNA polyplexes. CD-PEI was essentially nontoxic to HEK293 cells at concentrations optimal for gene delivery and mediated nearly 4-fold higher gene expression than unmodified PEI, which is relatively toxic to these cells. More importantly, addition of the pal-HI to CD-PEI enhanced gene expression by more than an order of magnitude compared to unmodified PEI, either with or without the pal-HI. Because of the relative ease with which CD-binding moieties may be attached to various types of ligands, CD-PEI may be a generally useful material for testing novel cell-specific targeting compounds.     

A bio-recognition device developed onto nano-crystals of carbonate apatite for cell-targeted gene delivery

The DNA delivery to mammalian cells is an essential tool for analyzing gene structure, regulation, and function. The approach holds great promise for the further development of gene therapy techniques and DNA vaccination strategies to treat and control diseases. Here, we report on the establishment of a cell-specific gene delivery and expression system by physical adsorption of a cell-recognition molecule on the nano-crystal surface of carbonate apatite. As a model, DNA/nano-particles were successfully coated with asialofetuin to facilitate uptake by hepatocyte-derived cell lines through the asialoglycoprotein receptor (ASGPr) and albumin to prevent non-specific interactions of the particles with cell-surface. The resulting composite particles with dual surface properties could accelerate DNA uptake and enhance expression to a notable extent. Nano-particles coated with transferrin in the same manner dramatically enhanced transgene expression in the corresponding receptor-bearing cells and thus our newly developed strategy represents a universal phenomenon for anchoring a bio-recognition macromolecule on the apatite crystal surface for targeted gene delivery, having immediate applications in basic research laboratories and great promise for gene therapy.