Gene Silencing Nucleic Acids Designed by Scanning Arrays: Anti-EGFR Activity of siRNA,Ribozyme and DNA Enzymes Targeting a Single Hybridization-accessible Region using the Same Delivery System

Gene silencing nucleic acids such as ribozymes, DNA enzymes (DNAzymes), antisense oligonucleotides (ODNs), and small interfering (si)RNA rely on hybridization to accessible sites within target mRNA for activity. However, the accurate prediction of hybridization accessible sites within mRNAs for design of effective gene silencing reagents has been problematic. Here we have evaluated the use of scanning arrays for the effective design of ribozymes, DNAzymes and siRNA sequences targeting the epidermal growth factor receptor (EGFR) mRNA. All three gene silencing nucleic acids designed to be complementary to the same array-defined hybridization accessible-site within EGFR mRNA were effective in inhibiting the growth of EGFR over-expressing A431 cancer cells in a dose dependent manner when delivered using the cationic lipid (Lipofectin) delivery system. Effects on cell growth were correlated in all cases with concomitant dose-dependent reduction in EGFR protein expression. The control sequences did not markedly alter cell growth or EGFR expression. The ribozyme and DNAzyme exhibited similar potency in inhibiting cell growth with IC₅₀ values of around 750 nM. In contrast, siRNA was significantly more potent with an IC₅₀ of about 100 nM when delivered with Lipofectin. The potency of siRNA was further enhanced when Oligofectamine was used to further improve both the cellular uptake and subcellular distribution of fluorescently labelled siRNA. Our studies show that active siRNAs can be designed using hybridization accessibility profiles on scanning arrays and that siRNAs targeting the same array-designed hybridization accessible site in EGFR mRNA and delivered using the same delivery system are more potent than ribozymes and DNAzymes in inhibiting EGFR expression in A431 cells.     

The efficiency of a novel delivery system (PEI600-Tat) in development of potent DNA vaccine using HPV16 E7 as a model antigen

DNA vaccination is a promising approach for inducing both humoral and cellular immune responses. The mode of plasmid DNA delivery is critical to make progress in DNA vaccination. Using human papillomavirus type 16 E7 as a model antigen, this study evaluated the effect of peptide-polymer hybrid including PEI600-Tat conjugate as a novel gene delivery system on the potency of antigen-specific immunity in mice model. At ratio of 10:50 PEI-Tat/E7DNA (w/w), both humoral and cellular immune responses were significantly enhanced as compared with E7DNA construct and induced Th1 response. Therefore, this new delivery system could have promising applications in gene therapy.     

Preparation and Characterization of Poly (D,L-Lactide-Co-Glycolide) Microspheres for Controlled Release of Poly(L-Lysine) Complexed Plasmid DNA

Purpose. To produce and characterize controlled release formulations of plasmid DNA (pDNA) loaded in poly (D,L-lactide-co-glycolide) (PLGA) microspheres both in free form and as a complex with poly (L-lysine). Methods. Poly (L-lysine) (PLL) was used to form pDNA/PLL complexes with complexation ratio of 1:0.125 and 1:0.333 w/w to enhance the stability of pDNA during microsphere preparation and protect pDNA from nuclease attack. pDNA structure, particle size, zeta potential, drug loading, in vitro release properties, and protection from DNase I were studied. Results. The microspheres were found to be spherical with average particle size of 3.1-3.5 m. Drug loading of 0.6% was targeted. Incorporation efficiencies of 35.1% and 29.4-30.6% were obtained for pDNA and pDNA/PLL loaded microspheres respectively. Overall, pDNA release kinetics following the initial burst did not correlate with blank microsphere polymer degradation profile suggesting that pDNA release is convective diffusion controlled. The percentage of supercoiled pDNA in the pDNA and pDNA/PLL loaded microspheres was 16.6 % and 76.7-85.6% respectively. Unencapsulated pDNA and pDNA/PLL degraded completely within 30 minutes upon the addition of DNase I. Encapsulation of DNA/PLL in PLGA microspheres protected pDNA from enzymatic degradation. Conclusions. The results show that using a novel process, pDNA can be stabilized and encapsulated in PLGA microspheres to protect pDNA from enzymatic degradation.     

Poly(l-lactide-co-glycolide) nanospheres conjugated with a nuclear localization signal for delivery of plasmid DNA

Polymeric nanospheres fabricated from biodegradable poly(lactide-co-glycolide) (PLGA) have been extensively investigated for applications in gene delivery. In this study, we show that the covalent conjugation of a nuclear localization signal (NLS, SV40 peptide) on PLGA nanospheres enhances the gene transfection efficiency. NLS conjugated PLGA copolymer was prepared by using a coupling reaction between maleimide-terminated PLGA copolymer and NLS in the presence of Imject maleimide conjugation buffer. PLGA nanospheres encapsulating plasmid (pDNA) were prepared by using a double emulsion-solvent evaporation method. The kinetics of in vitro release of pDNA from PLGA nanospheres was determined with UV in phosphate buffered saline (PBS). Gene transfection efficiency in human dermal fibroblasts was tested in vitro using nanospheres encapsulating the luciferase gene. The conjugation of the NLS peptide to the PLGA nanospheres could improve the nuclear localization and/or cellular uptake of PLGA nanosphere/pDNA constructs and thereby improve the transfection efficiency of a PLGA nanosphere gene delivery system. The pDNA was released from PLGA nanospheres over nine days. NLS conjugation enhanced the gene transfection efficiency in vitro by 1.2 ～ 3.2-fold over 13 days. PLGA/pDNA nanospheres appeared to be superior to PEI/pDNA complexes for the long-term expression of pDNA. Furthermore, the level of the sustained gene expression of the PLGA nanospheres was enhanced by the conjugation of NLS to the PLGA nanospheres. This study showed that the NLS conjugation enhanced the gene transfection efficiency of the PLGA nanosphere gene delivery system in vitro and that the enhanced gene expression was sustained for at least 13 days.     

Structure Effect of Some New Anticancer Pt(II) Complexes of Amino Acid Derivatives with Small Branched or Linear Hydrocarbon Chains on Their DNA Interaction

The aim of this study was to investigate the structure effect and identify the modes of binding of amino acid‐Pt complexes to DNA molecule for cancer treatment. Hence, three novel water soluble platinum complexes, [Pt(phen)(R‐gly)]NO₃ (where phen is 1,10‐phenanthroline, R‐gly is methyl, amyl, and isopentyl‐glycine), have been synthesized and characterized by spectroscopic methods, conductivity measurements, and chemical analysis. The anticancer activities of synthesized complexes were investigated against human breast cancer cell line of MDA‐MB 231. The 50% cytotoxic concentration values were determined to be 42.5, 58, and 70 μm for methyl‐, amyl‐, and isopentyl‐gly complexes, respectively. These complexes were interacted with calf thymus DNA (ct‐DNA) via positive cooperative interaction. The modes of binding of the complexes to DNA were investigated by fluorescence spectroscopy and circular dichroism in combination with a molecular docking study. The result indicates that complexes with small or branched hydrocarbon chains can intercalate with DNA. This is while amyl complexes with linear chains interacted additionally via groove binding. The results of the negative value of Gibbs energy for binding of isopentyl‐platinum to DNA and those of the molecular docking were coherent. Furthermore, the docking results demonstrated that hydrophobic interaction plays an important role in the complex–DNA interaction.     

Effect of a Chemical Delivery System for Dexamethasone (Dex-CDS) on Peritumoral Edema in an Experimental Brain Tumor Model

Pharmaceutical Research -     

镍致DNA链断裂及对聚腺苷二磷酸核糖聚酶活性的影响

目的 探讨镍所致DNA链断裂及其所诱导的聚腺苷二磷酸核糖聚酶（PARP）之间的关系。方法 培养猴肾Vero细胞,分别用0、125、250、500、1000μmol/L的醋酸镍染毒2.5、6和12h;苔盼蓝计数法检测细胞存活率。单细胞凝胶电泳法检测DNA链断裂,^3H掺入法检测PARP活性。结果 镍浓度与DNA链断裂程度之间存在剂量-效应和时间-效应关系;镍也可诱导PARP活性显著升高,但在高浓度和长时间染毒时,其活性不再增加,结论 镍所致的DNA链断裂与其诱导的PARP活性之间存在特殊关系。这可能与镍的致癌性相关。     

Molecular dosimetry of DNA adducts in rainbow trout (Oncorhynchus mykiss) exposed to benzo(a)pyrene by different routes

 Farm raised rainbow trout (Oncorhynchus mykiss) were exposed by various routes to benzo(a)pyrene (BP) as a representative carcinogenic polycyclic aromatic hydrocarbon (PAH). Following exposure of fish to the chemical by intraperitoneal (i.p.) injection, ³²P-postlabelling studies indicated that non-feral trout were relatively resistant to the formation of BP-DNA adducts in liver. No adducts were detected in fish exposed to single doses (20 mg/kg) of BP. Multiple exposures (e.g. 2×25 mg/kg) were necessary in order for adducts to be detected, indicating that induction of the metabolising enzymes required for the bioactivation of BP is necessary. These studies provided reference information on DNA adducts for comparison with data from subsequent experiments at environmentally realistic low level exposures. Two types of low level aquatic exposure were carried out. The first procedure exposed fish for 30 days to a nominally constant low level (1.2 and 0.4 μg/l) of a homogeneous dispersion of BP in water, to simulate low level aquatic environmental exposures. Following ³²P-postlabelling analysis of the liver DNA of exposed fish, BP-DNA adducts were not detected. In the second procedure, fish were exposed to a constant low level of BP (ca. 0.5 μg/l) for 15 days then to a pulse (60 μg/l) which was allowed to naturally decline (to ca. 2 μg/l) during a further 15 days. Following this exposure, significant levels of BP-DNA adducts were detected in livers of trout. The effect of dietary exposures was investigated by feeding trout a diet containing either 58 μg or 288 μg BP per day for 6 days, equivalent to total doses of 43 mg/kg and 216 mg/kg. In both cases BP-DNA adducts were detected in livers of exposed fish. The results provide useful information on the types of exposures to PAHs which may pose a genotoxic risk to fish in the environment. Received: 5 April 1994 / Accepted: 31 May 1994     

Elevated levels of DNA repair enzymes and antioxidative enzymes by ( )-catechin in murine microglia cells after oxidative stress

(+)-Catechin possesses a broad range of pharmacological properties, including antioxidative effect. However, little is reported on the mechanism by which (+)-catechin protects microglia cells from DNA damage by oxidative stress. In this study, TUNEL assay and DNA electrophorysis indicated that (+)-catechin markedly blocked DNA fragmentation and apoptosis of microglia cells by tBHP exposure. A potent antioxidative effect of (+)-catechin was confirmed by comparison with a putative antioxidant agent, N-acetylcysteine at the lower doses. Furthermore, the increased intracellular ROS by tBHP exposure were scavenged by elevated activities of catalase (CAT) and superoxide dismutase (SOD) after (+)-catechin treament. (+)-Catechin partially inhibited the activation of caspase-3, thereby both cleavage of poly (ADP-ribose) polymerase (PARP) and degradation of inhibitor of caspase-activated DNase (ICAD) were effectively abolished. In addition, the expression of PARP for repair of impaired DNA was significantly increased by (+)-catechin treatment. Taken together, these data suggest that protective effects of (+)-catechin against oxidative DNA damage of microglia cells is exerted by the increased expression of DNA repair enzyme PARP and antioxidant enzyme activities.     

Analysis of Micelle Formation of an Adriamycin-Conjugated Poly(Ethylene Glycol)–Poly(Aspartic Acid) Block Copolymer by Gel Permeation Chromatography

The micelle-forming behavior of a drug–block copolymer conjugate adriamycm-conjugated poly(ethylene glycol)–poly(aspartic acid) block copolymer; PEG-P[Asp(ADR)] was analyzed by gel permeation chromatography (GPC). Four compositions of the conjugates were observed to form micellar structures in aqueous media, and their micelle-forming behavior was found to be dependent on the composition and media. These micelles did not reach equilibrium within short time periods like low molecular weight surfactants. One composition formed stable micelles in the presence of serum.     

DNA damage induced by shikonin in the presence of Cu(II) ions: potential mechanism of its activity to apoptotic cell death

Shikonin (β-alkannin), a naphthazarin derivative, has shown a variety of abilities such as anti-inflammatory, antitumoral, cytotoxic, and antimicrobial activities. In the presence of Cu(II), shikonin caused breakage of supercoiled plasmid pBR322 DNA. Other metal ions tested [Mg(II), Ca(II), and Ni(II)] were ineffective and only Fe(II) has the same ability in the DNA breakage reaction. The involvement of active oxygen in the reaction was established by the inhibition of DNA breakage by superoxide dismutase, catalase, thiourea, sodium azide, potassium iodide, and sodium benzoate. Cu(I) was shown to be an essential intermediate using the Cu(I)-specific sequestering reagent neocuproine. Shikonin induced HeLa cell apoptosis involved in the mechanism of increasing intracellular reactive oxygen species (ROS). It was suggested that shikonin generated ROS as a pro-oxidant in the presence of Cu(II), and ROS resulted in DNA damage and apoptotic cell death in cells.     

Analysis of cell cycle regulation by 1-mono-O-acyl-3-O-(alpha-D-sulfoquinovosyl)-glyceride (SQMG), an inhibitor of eukaryotic DNA polymerases

One of the sulfo-lipids, 1-mono-O-acyl-3-O-(alpha-D-sulfoquinovosyl)-glyceride (SQMG), potently and selectively inhibited the activity of mammalian DNA polymerases. SQMG was also a potent apoptosis inducer and the SQMG effect occurred through the induction of G1 arrest with a reduction in the proportion of cells in the S phase. SQMG clearly increased the levels of p53 and p21 proteins, but did not induce the expression of p27 and p16 proteins. SQMG markedly reduced the pRb protein level and inhibited pRb phosphorylation after 48hr. These results suggested that SQMG activates the G1 checkpoint as a result of the DNA polymerase inhibition, and then promotes a p53-dependent apoptotic response. Since aphidicolin, a well-known replicative DNA polymerase inhibitor, did not promote these protein expressions, the apoptosis-inducing pathway by SQMG differs from that by aphidicolin.     

Reduced camptothecin sensitivity of estrogen receptor‐positive human breast cancer cells following exposure to di(2‐ethylhexyl)phthalate (DEHP) is associated with DNA methylation changes

Di(2‐ethylhexyl)phthalate (DEHP) has been considered as an estrogen receptor alpha (ERα) agonist due to its ability to interact with ERα and promote the cell proliferation of ERα‐positive breast cancer cells. The impact of DEHP on the chemical therapy in breast cancer is little known. Two breast cancer cell lines, MCF‐7 (ERα‐dependent) and MDA‐MB‐231 (ERα‐independent) were examined. We found that DEHP impaired the effectiveness of camptothecin (CPT) and alleviated the CPT‐induced formation of reactive oxygen species in ERα‐positive MCF‐7 cells, but not in ERα‐negative MDA‐MB‐231 cells. DEHP also significantly protected MCF‐7 cells against the genotoxicity of CPT. Genome‐wide DNA methylation profiling revealed that after 48 hours of exposure to 100 μM DEHP, MCF‐7 cells exhibited a significant change in their DNA methylation pattern, including hypermethylation of 700 genes and hypomethylation of 221 genes. The impaired therapeutic response to CPT in DEHP‐exposed MCF‐7 cells is probably mediated by epigenetic changes, especially through Wnt/β‐catenin signaling. A zebrafish xenograft model confirmed the disruptive effect of DEHP on CPT‐induced anti‐growth of MCF‐7 cells. In summary, DEHP exposure induces acquired CPT‐resistance in breast cancer cells and epigenetic changes associated with Wnt/β‐catenin signaling activation are probably depending on an ER‐positive status.     

Transport Alteration of 4-Phenyl Butyric Acid Mediated by a Sodium- and Proton-Coupled Monocarboxylic Acid Transporter System in ALS Model Cell Lines (NSC-34) Under Inflammatory States

4-Phenyl butyric acid (PBA) has histone deacetylase inhibitory and neuroprotective effects. We aimed to examine the transport alteration activity of PBA in control (WT) and disease (MT) model cell lines of an amyotrophic lateral sclerosis (ALS) model. The transport characteristics of PBA were examined uptake rates and mRNA expression levels in NSC-34 cell lines. PBA uptake was pH, sodium, and concentration dependent. The K_m and V_max values for PBA uptake in the MT were more than two-fold higher than those in the WT. The presence of monocarboxylic acids (MA) and inhibitors of MA transporter (MCT) inhibited the uptake of PBA. PBA showed competitive inhibition in the presence of MAs in both cell lines. SiRNA transfection studies showed that PBA can be transported to NSC-34 cell lines through sodium-coupled MCT1. TNF-α and H₂O₂ increased, but LPS and glutamate reduced the uptake rate after the pretreatment of the MT cell lines. SMCT1 mRNA expression levels, in the presence of oxidative stress inducing agents, showed consistent results with the uptake results. These results demonstrate that PBA can be transported to the ALS model NSC-34 cell lines by sodium- and proton-coupled MCTs, and MA plays a vital role in the prevention of neurodegenerative diseases.     

Evaluation of cytotoxicity and oxidative DNA damaging effects of di(2-ethylhexyl)-phthalate (DEHP) and mono(2-ethylhexyl)-phthalate (MEHP) on MA-10 Leydig cells and protection by selenium

Di(2-ethylhexyl)-phthalate (DEHP) is the most abundantly used phthalate derivative, inevitable environmental exposure of which is suspected to contribute to the increasing incidence of testicular dysgenesis syndrome in humans. Oxidative stress and mitochondrial dysfunction in germ cells are suggested to contribute to phthalate-induced disruption of spermatogenesis in rodents, and Leydig cells are one of the main targets of phthalates’ testicular toxicity. Selenium is known to be involved in the modulation of intracellular redox equilibrium, and plays a critical role in testis, sperm, and reproduction. This study was aimed to investigate the oxidative stress potential of DEHP and its consequences in testicular cells, and examine the possible protective effects of selenium using the MA-10 mouse Leydig tumor cell line as a model. In the presence and absence of selenium compounds [30 nM sodium selenite (SS), and 10 μM selenomethionine (SM)], the effects of exposure to DEHP and its main metabolite mono(2-ethylhexyl)-phthalate (MEHP) on the cell viability, enzymatic and non-enzymatic antioxidant status, ROS production, p53 expression, and DNA damage by alkaline Comet assay were investigated. The overall results of this study demonstrated the cytotoxicity and genotoxicity potential of DEHP, where MEHP was found to be more potent than the parent compound. SS and SM produced almost the same level of protection against antioxidant status modifying effects, ROS and p53 inducing potentials, and DNA damaging effects of the two phthalate derivatives. It was thus shown that DEHP produced oxidative stress in MA-10 cells, and selenium supplementation appeared to be an effective redox regulator in the experimental conditions used in this study, emphasizing the critical importance of the appropriate selenium status.     

Establishment of an Evaluation Method for Gene Silencing by Serial Pulmonary Administration of siRNA and pDNA Powders: Naked siRNA Inhalation Powder Suppresses Luciferase Gene Expression in the Lung

In order to evaluate the in vivo effect of inhaled formulations, it is a gold standard to create a lung metastasis model by intravenously injecting cancer cells into an animal. Because the cancer grows from the blood vessel side, there is a possibility of underestimating the effect of an inhaled formulation administered to the lung epithelium side. In addition, the metastasis model has disadvantages in terms of preparation time and expense. The present study aimed to establish a new method to evaluate the effect of an inhaled small interfering RNA (siRNA) formulation that is more correct, more rapid, and less expensive. We investigated whether siRNA can suppress gene expression of plasmid DNA (pDNA) by serial pulmonary administration of siRNA and pDNA powders prepared by spray-freeze-drying. We revealed that formulations of dry siRNA powder significantly suppressed gene expression of pDNA powder compared with a control group with no siRNA. Naked siRNA inhalation powder with no vector showed the suppression of gene expression equivalent to that of an siRNA-polyethyleneimine complex without damaging tissues. These results show that the present method is suitable for evaluating the gene-silencing effect of inhaled siRNA powders.     

Synthesis of bisphosphonate derivatives of ATP by T4 DNA ligase, ubiquitin activating enzyme (E1) and other ligases

T4 DNA ligase and the ubiquitin activating enzyme (E1), catalyze the synthesis of ATP beta,gamma-bisphosphonate derivatives. Concerning T4 DNA ligase: (i) etidronate (pC(OH)(CH(3))p) displaced the AMP moiety of the complex E-AMP in a concentration dependent manner; (ii) the K(m) values and the rate of synthesis k(cat) (s(-1)), determined for the following compounds were, respectively: etidronate, 0.73+/-0.09 mM and (70+/-10)x10(-3) s(-1); clodronate (pCCl(2)p), 0.08+/-0.01 mM and (4.1+/-0.3)x10(-3) s(-1); methylenebisphosphonate (pCH(2)p), 0.024+/-0.001 mM and (0.6+/-0.1)x10(-3) s(-1); tripolyphosphate (P(3)) (in the synthesis of adenosine 5'-tetraphosphate, p(4)A), 1.30+/-0.30 mM and (6.2+/-1.1)x10(-3) s(-1); (iii) in the presence of GTP and ATP, inhibition of the synthesis of Ap(4)G was observed with clodronate but not with pamidronate (pC(OH)(CH(2)-CH(2)-NH(3))p). Concerning the ubiquitin activating enzyme (E1): methylenebisphosphonate was the only bisphosphonate, out of the ones tested, that served as substrate for the synthesis of an ATP derivative (K(m)=0.36+/-0.09 mM and k(cat)=0.15+/-0.02 s(-1)). None of the above bisphosphonates were substrates of the reaction catalyzed by luciferase or by acyl-CoA synthetase. The ability of acetyl-CoA synthetase to use methylenebisphosphonate as substrate depended on the commercial source of the enzyme. In our view this report widens our knowledge of the enzymes able to metabolize bisphosphonates, a therapeutic tool widely used in the treatment of osteoporosis.     

DNA interactions of new cytotoxic tetrafunctional dinuclear platinum complex trans,trans-[{PtCl2(NH3)}2(piperazine)

A new tetrafunctional dinuclear platinum complex trans,trans-[{PtCl2(NH3)}2(piperazine)] with sterically rigid linking group was designed, synthesized and characterized. In this novel molecule, the DNA-binding features of two classes of the platinum compounds with proven antitumor activity are combined, namely trans oriented bifunctional mononuclear platinum complexes with a heterocyclic ligand and polynuclear platinum complexes. DNA-binding mode of this new complex was analyzed by various methods of molecular biology and biophysics. The complex coordinates DNA in a unique way and interstrand and intrastrand cross-links are the predominant lesions formed in DNA in cell-free media and in absence of proteins. An intriguing aspect of trans,trans-[{PtCl2(NH3)}2(piperazine)] is that, using a semi-rigid linker, interstrand cross-linking is diminished relative to other dinuclear platinum complexes with flexible linking groups and lesions that span several base pairs, such as tri- and tetrafunctional adducts, become unlikely. In addition, in contrast to the inability of trans,trans-[{PtCl2(NH3)}2(piperazine)] to cross-link two DNA duplexes, the results of the present work convincingly demonstrate that this dinuclear platinum complex forms specific DNA lesions which can efficiently cross-link proteins to DNA. The results substantiate the view that trans,trans-[{PtCl2(NH3)}2(piperazine)] or its analogues could be used as a tool for studies of DNA properties and their interactions or as a potential antitumor agent. The latter view is also corroborated by the observation that trans,trans-[{PtCl2(NH3)}2(piperazine)] is a more effective cytotoxic agent than cisplatin against human tumor ovarian cell lines.     

Pulmonary delivery of siRNA against acute lung injury/acute respiratory distress syndrome

The use of small interfering RNAs (siRNAs) has been under investigation for the treatment of several unmet medical needs, including acute lung injury/acute respiratory distress syndrome (ALI/ARDS) wherein siRNA may be implemented to modify the expression of pro-inflammatory cytokines and chemokines at the mRNA level. The properties such as clear anatomy, accessibility, and relatively low enzyme activity make the lung a good target for local siRNA therapy. However, the translation of siRNA is restricted by the inefficient delivery of siRNA therapeutics to the target cells due to the properties of naked siRNA. Thus, this review will focus on the various delivery systems that can be used and the different barriers that need to be surmounted for the development of stable inhalable siRNA formulations for human use before siRNA therapeutics for ALI/ARDS become available in the clinic.