Yongyut Rojanasakul

Reactive Oxygen Species Regulate Angiogenesis and Tumor Growth through Vascular Endothelial Growth Factor

Reactive oxygen species (ROS) are associated with multiple cellular functions such as cell proliferation, differentiation, and apoptosis. However, the direct roles of endogenous ROS production still remain to be elucidated. In this study, we found that high levels of ROS were spontaneously produced by ovarian and prostate cancer cells. This elevated ROS production was inhibited by NADPH oxidase inhibitor diphenylene iodonium (DPI) and mitochondria electron chain inhibitor rotenone in the cells. To further analyze the source of ROS production, we found that ovarian cancer cells have much higher expression of NOX4 NADPH oxidase, and that specific inhibition of NADPH oxidase subunit p47(phox) diminished ROS production. To analyze the functional relevance of ROS production, we showed that ROS regulated hypoxia-inducible factor 1 (HIF-1) and vascular endothelial growth factor (VEGF) expression in ovarian cancer cells. Elevated levels of endogenous ROS were required for inducing angiogenesis and tumor growth. NOX4 knockdown in ovarian cancer cells decreased the levels of VEGF and HIF-1 alpha and tumor angiogenesis. This study suggests a new mechanism of higher ROS production in ovarian cancer cells and provides strong evidence that endogenous ROS play an important role for cancer cells to induce angiogenesis and tumor growth. This information may be useful to understand the new mechanism of cancer cells in inducing tumorigenesis and to develop new therapeutic strategy by targeting ROS signaling in human cancer in the future.

The Transport Barrier of Epithelia: A Comparative Study on Membrane Permeability and Charge Selectivity in the Rabbit

The transport barrier of the epithelia presents one of the major problems limiting the effective use of these tissues as alternate delivery routes for macromolecules such as peptides and proteins. In the present study, two membrane transport properties, namely, the permeability and permselectivity of the shunt pathway, were investigated and compared in various tissues including the nasal, tracheal, bronchial, buccal, rectal, vaginal, corneal, epidermal, duodenal, jejunal, ileal, and colonic epithelia. Membrane permeability was evaluated using a combined method based on electrical conductance and flux measurements of a hydrophilic fluorescent probe, 6-carboxy fluorescein (CF). Membrane permselectivity or the charge discriminating ability of the membrane was evaluated by KCl diffusion potential measurements. The results indicate that all epithelia under investigation possess a relatively high degree of permeation barrier and are highly selective for the absorption of positively charged solutes. Shunt path permeability was found to vary greatly among tissues from different epithelia, whereas membrane charge selectivity was relatively constant in these tissues. A good correlation was observed between membrane electrical conductance and steady-state flux of CF, indicating a paracellular transport of the compound. The rank order of the intrinsic membrane permeability was as follows: intestinal≈ nasal ≥ bronchial ≥ tracheal > vaginal ≥ rectal > corneal > buccal > skin. Membrane permselectivity, expressed as the ratio of transport number (positive over negative), ranges from 1.78 for the buccal to 1.33 for the rectal epithelium. These results suggest that, for effective delivery purposes, permeation enhancing methods, by either increasing tissue permeability or modifying drug-membrane charge selectivity, are generally required. The permeation data also suggest that the respiratory epithelia represent good alternate routes for drug delivery, particularly for those that are orally ineffective, i.e., due to extensive gastrointestinal tract degradation or first-pass metabolism.

Vanadate Induces p53 Transactivation through Hydrogen Peroxide and Causes Apoptosis

Vanadium is a metal widely distributed in the environment. Although vanadate-containing compounds exert potent toxic effects on a wide variety of biological systems, the mechanisms controlling vanadate-induced adverse effects remain to be elucidated. The present study investigated the vanadate-induced p53 activation and involvement of reactive oxygen species (ROS) in p53 activation as well as the role of p53 in apoptosis induction by vanadate. Exposure of mouse epidermal JB6 cells to vanadate led to transactivation of p53 activity in a time- and dose-dependent manner. It also caused mitochondrial damage, apoptosis, and generated ROS. Scavenging of vanadate-induced H2O2 byN-acetyl-l-cysteine (a general antioxidant) or catalase (a specific H2O2 inhibitor), or the chelation of vanadate by deferoxamine, resulted in inhibition of p53 activation and cell mitochondrial damage. In contract, an increase in H2O2 generation in response to superoxide dismutase or NADPH enhanced these effects caused by vanadate. Furthermore, vanadate-induced apoptosis occurred in cells expressing wild-type p53 (p53+/+) but was very weak in p53-deficient (p53−/−) cells. These results demonstrate that vanadate induces p53 activation mainly through H2O2 generation, and this activation is required for vanadate-induced apoptosis.

Antisense oligonucleotide therapeutics : drug delivery and targeting

Abstract Oligonucleotide(ON)-based therapy, although in an early stage of development, promises to provide new and highly specific tools for the treatment of human diseases such as virus-associated illnesses and cancers. Like gene therapy, ON therapy is a rapidly growing field with great therapeutic potential. However, the two types of therapy differ fundamentally in their approach. In gene therapy, missing or defective genes are added or replaced with functional versions, while in ON therapy, existing but abnormally expressed genes are inhibited. In this article, we will focus on ON therapy with an emphasis on issues related to ON drug delivery, stability, and targeting. ONs have several advantages over traditional drugs, notably their exquisite specificity to target sites and their ease of design. However, their effective use has been limited due to several problems. For example, naturally occuring ONs contain phosphodiester backbones that are easily degraded in a biological environment and therefore must be protected or modified to render stability. In addition, because of their large molecular size and charge, these compounds are poorly taken up by cells and therefore may not reach their target site. Moreover, problems associated with cellular targeting, potential toxicity, and affinity of ONs to the target sites pose major challenges to the successful utilization of these compounds. Here we shall examine recent findings, relative advantages and disadvantages of various ON delivery methods, as well as the common pitfalls peculiar to each strategy.

Regulation of tight junction permeability by calcium mediators and cell cytoskeleton in rabbit tracheal epithelium

The present study investigates the mechanisms controlling tight junction permeability of the tracheal epithelium, with an emphasis on the regulatory role of intra- and extracellular calcium as well as the cell cytoskeleton. The tracheas were isolated from rabbits and their junctional permeability barrier was investigated in vitro by means of transepithelial electrical resistance measurements and flux measurements of the radiolabeled paracellular tracer, 14C-mannitol. The effects of intra- and extracellular calcium were studied using the calcium ionophore A 23187 and EGTA, and that of the cytoskeleton was investigated using cytochalasin B. Intracellular calcium of the tracheal epithelium was monitored microfluorometrically using the specific calcium indicator, Fura-2 AM (acetoxymethyl ester). The results indicate that the tight junction permeability of the trachea was significantly increased upon treatment with all three of the test compounds, as evidenced by a substantial decrease in transepithelial electrical resistance and an increase in transepithelial flux of 14C-mannitol. The effects of EGTA and cytochalasin B on the tight junction permeability are fully reversible upon removal of the compounds from the bathing media. On the other hand, tissues treated with the calcium ionophore demonstrate a partial or no recovery in membrane permeability, depending on the intracellular calcium levels. Moderate and transient increases in intracellular calcium caused a partial reversibility of the membrane resistance, while high and sustained intracellular calcium levels induce a complete irreversibility of the membrane resistance. These results suggest that high extracellular calcium levels and low intracellular calcium levels are required for the normal maintenance of the junctional permeability in the tracheal epithelium. Studies using cytochalasin B indicate that there is also a close relationship between the tight junctions and the organization of actin microfilaments. Alterations of these structures as well as cellular calcium levels can result in a substantial change in transepithelial permeability. Therefore compounds that affect tight junction permeability may exert their action through the calcium and cytoskeleton mechanisms.

Targeted gene delivery to alveolar macrophages via Fc receptor-mediated endocytosis

Alveolar macrophage (AM) plays important roles in lung homeostasis and pathogenesis of diseases. The study of macrophage gene function and regulation as well as its potential therapeutic intervention will require the development of vectors capable of safe and efficient transfer of DNA to the AM. In the present study, we report a new transfection system that utilizes Fc receptor-mediated endocytosis as a means to target DNA to the AM. This system employs molecular conjugates consisting of a cognate moiety, in this case IgG which recognizes the AM Fc receptor, covalently-linked to a DNA-binding moiety, such as a cationic polyamine. A Complex was formed between immunoglobulin G-polylysine conjugate (IgG-pL) and plasmid DNA carrying the LacZ reporter gene (pSVβ). The conjugate-DNA complex was added directly to the AMs in culture and incubated for 24 h, after which LacZ gene expression was analyzed for β-galactosidase activity by microfluorometry using a fluorogenic β-galactosidase substrate, 5-dodecanoylaminofluorescein di-β-D-galactopyranoside (C12FDG). The AMs treated with the IgG-pL/DNA complex exhibited galactosidase activity significantly augmented over background levels. Effective gene transfer was shown to require both the DNA-binding moiety and cognate moiety for the cell surface receptor. Specific internalization of the complex by the Fc receptor pathway was verified by competitive inhibition using excess IgG. Under this condition, LacZ gene expression was inhibited, suggesting complex internalization through the Fc mediated endocytosis pathway. The requirement of Fc receptors for complex internalization was further demonstrated using cells that lack Fc receptors, e.g., alveolar epithelial cells. When exposed to the IgG-pL/pSVβ complex, these epithelial cells showed no susceptibility to gene transfer. Thus, the immune conjugate system may be used to accomplish targeted gene delivery to the AMs via the endocytosis pathway. Finally, the conjugate system was found to be nontoxic at concentrations effectively enhancing gene transfer, thereby, suggesting its potential safety in vivo.

Receptor-Mediated Peptide Delivery in Pulmonary Epithelial Monolayers

The present study investigated the feasibility of utilizing receptor-mediated endocytosis as a means to enhance peptide delivery to the pulmonary epithelium. The strategy employs a molecular conjugate consisting of a cognate moiety, transferrin (TF), covalently-linked to a model polypeptide, horseradish peroxidase (HRP), via a reversible disulfide linkage. A cultured alveolar epithelial monolayer system was used to simulate the conditions of the pulmonary epithelium and to allow accurate quantitation of intra- and transcellular peroxidase transport. The alveolar cells were isolated from rat lungs by enzymatic digestion and grown on microporous tissue culture-treated polycarbonate filters. A significant increase in the uptake of HRP by the cell monolayer was observed upon its conjugation with TF. The effect was found to be concentration-dependent, being more pronounced at low concentrations, i.e., 3.9- and 1.2-fold increase over unconjugated HRP controls at the concentration levels of 0.05 and 1.50 U/ml respectively. Effective peroxidase uptake was shown to require the TF cognate moiety for the cell surface receptor. Specific internalization of the conjugate by the TF endocytic pathway was verified by competition for the TF receptor. Conjugate internalization was not followed by a proportional increase in transcytosis, i.e., at 0.05 U/ml conjugate level, a 1.7-fold increase in transcytosis was observed as compared to 3.9-fold for endocytosis. Effective enhancement of transcytosis was achieved by treating the monolayers with brefeldin A (BFA), a compound known to affect intracellular transport of TF receptor complexes. At 1.6 µ/ml concentration level, BFA promoted a >20-fold increase in the rate of transcytosis of the conjugate in both the apical-to-basal and basal-to-apical directions. This effect was not associated with membrane leakage since BFA-treated monolayers maintained tight barrier to transport of the paracellular permeability solute 14C mannitol. In addition, BFA had no significant effect on the transport of free HRP. Instead, the effect of BFA on conjugate transport was mediated by TF receptors since excess free TF competitively inhibited transcytosis of the conjugate. Thus, our results are consistent with the TF receptor-mediated transport of the conjugate and its enhancement through the intracellular rerouting of the conjugate by BFA. The findings in this study may potentially be relevant to the design of drug delivery systems that can enhance intra- or transcellular uptake of therapeutic peptides in the pulmonary epithelium.

One-electron reduction of vanadate by ascorbate and related free radical generation at physiological pH

The one-electron reduction of vanadate (vanadium(V)) by ascorbate and related free radical generation at physiological pH was investigated by ESR and ESR spin trapping. The spin trap used was 5,5-dimethyl-1-pyrroline N-oxide (DMPO). Incubation of vanadium(V) with ascorbate generated significant amounts of vanadium(IV) in phosphate buffer (pH 7.4) but not in sodium cacodylate buffer (pH 7.4) nor in water. The vanadium(IV) yield increased with increasing ascorbate concentration, reaching a maximum at a vanadium(V): ascorbate ratio of 2:1. Addition of formate to the incubation mixture containing vanadium(V), ascorbate, and phosphate generated carboxylate radical (.COO-), indicating the formation of reactive species in the vanadium(V) reduction mechanism. In the presence of H2O2 a mixture of vanadium(V), ascorbate, and phosphate buffer generated hydroxyl radical (.OH) via a Fenton-like reaction (vanadium(IV)+H2O2-->vanadium(V)+.OH+OH-). The .OH yield was favored at relatively low ascorbate concentrations. Omission of phosphate sharply reduced the .OH yield. The vanadium(IV) generated by ascorbate reduction of vanadium(V) in the presence of phosphate was also capable of generating lipid hydroperoxide-derived free radicals from cumene hydroperoxide, a model lipid hydroperoxide. Because of the ubiquitous presence of ascorbate in cellular system at relatively high concentrations, one-electron reduction of vanadium(V) by ascorbate together with phosphate may represent an important vanadium(V) reduction pathway in vivo. The resulting reactive species generated by vanadium(IV) from H2O2 and lipid hydroperoxide via a Fenton-like reaction may play a significant role in the mechanism of vanadium(V)-induced cellular injury.

Generation of thiyl and ascorbyl radicals in the reaction of peroxynitrite with thiols and ascorbate at physiological pH

Electron spin resonance (ESR) spin trapping was utilized to investigate the reaction of peroxynitrite with thiols and ascorbate at physiological pH. The spin trap used was 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The reaction of peroxynitrite with DMPO generated 5,5-dimethylpyrrolidone-(2)-oxy-(1) (DMPOX). Formate enhanced the peroxynitrite decomposition but did not generate any detectable amount of formate-derived free radicals. Thus, the spin trapping measurements provided no evidence for hydroxyl (.OH) radical generation in peroxynitrite decomposition at physiological pH. Thiols (glutathione, cysteine, and penicillamine) and ascorbate reacted with peroxynitrite to generate the corresponding thiyl and ascorbyl radicals. The one-electron oxidation of thiols by peroxynitrite may be one of the important mechanisms for peroxynitrite-induced toxicity and ascorbate may provide a detoxification pathway.

Alveolar Permeability Enhancement by Oleic Acid and Related Fatty Acids: Evidence for a Calcium-Dependent Mechanism

Pulmonary exposure to oleic acid (OA) is associated with permeability alterations and cellular damage; however, the exact relationship between these two effects has not been clearly established. Using cultured alveolar epithelial monolayers, we demonstrated that OA and some other fatty acids (≤50 µM) can induce permeability changes without detectable cellular damage. At higher concentrations, however, OA caused severe membrane damage and leakage to solute flux. The permeability enhancing effect of OA was observed with both the paracellular marker 3H-mannitol and the lipophilic transcellular indicator 14C-progesterone. While the effect of OA on transcellular permeability may be attributed to its known effect on membrane fluidity, the paracellular promoting effect of OA and its mechanism are not well established. We postulated that OA may increase paracellular permeability through a Ca2+-dependent tight junction mechanism. Using dual-excitation fluorescence microscopy, we demonstrated that OA can increase intracellular calcium, [Ca2+]i , in a dose-dependent manner. This effect was transient at low OA concentrations (≤50 µM) but became more pronounced and sustained at higher concentrations. Free hydroxyl and unsaturated groups were required for this activation since esterified OA (oleic methyl ester) and stearic acid (a saturated fatty acid with equal chain length) had much reduced effects on both the [Ca2+]i and the permeability alterations. Degree of unsaturation was unimportant since linolenic acid (18:3), linoleic acid (18:2), and OA (18:1) had similar and comparable effects on the two parameters. When the alveolar epithelium was bathed with Ca2+ -free medium, OA failed to elevate [Ca2+ ]i , suggesting that Ca2+ influx from the extracellular medium is responsible for the observed [Ca2+]i rise. This effect of OA was not due to nonspecific membrane damage since the monolayer maintained its integrity and the [Ca2+]ireturned to pretreatment levels after an initial transient rise. Moreover, the permeability alteration was fully reversible upon removal of OA. These results suggest that the alveolar permeability may be reversibly enhanced by sublethal concentrations of oleic acid.