Reversion of the multiple-drug resistance phenotype mediated by short interfering RNAs

Repression of MDR1 gene expression and restoration of cancer cell sensitivity to cytostatic agents were studied with the use of synthetic siRNAs directed to different MDR1 mRNA regions. Short interfering RNAs that enhance sensitivity of drug-resistant cells to vinblastine were revealed. The ability of various siRNAs to repress gene expression does not correlate with the efficiency of antisense oligonucleotides complementary to the same mRNA regions. An siRNA sample remained intact in a medium during a period of time sufficient to exhibit biological activity. The results of our study suggest that siRNAs can be considered as a basis for therapeutic drugs for enhancement of the efficiency of antitumor chemotherapy. __________ Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 5, pp. 1260–1267, May, 2005.     

10-23 DNA enzymes for targeting MDR1 mRNA

The properties of the 31-mer 10-23 DNA enzyme and its analog with the terminal 3"-3" internucleotide linkage, which are complementary to an mRNA region of the multi-drug resistance gene MDR1, were investigated. DNA enzymes can selectively cleave RNA with high efficiency in a catalytic mode as exemplified by a synthetic 18-mer fragment of MDR1 mRNA.     

基于报告基因技术的MDR1转录抑制剂高通量筛选模型的建立和应用

MDR1基因是引起肿瘤多药耐药的主要基因,其编码的P-gp蛋白可持续将药物由胞内排出胞外以降低胞内药物浓度导致多药耐药,MDR1基因的转录抑制剂可抑制MDR1基因在癌细胞中的表达,从而逆转肿瘤多药耐药.通过克隆MDR1基因的启动子,将其插入pGL3-basic质粒构建MDR1-luc＋报告基因载体,再将重组载体转染入HepG2肝癌细胞并筛选单克隆细胞株,构建了MDR1启动子的高通量筛选模型,Z′因子为0.75;通过对中药样品库的筛选,得到两种中药提取物高良姜水提物、红豆蔻醇提物有明显耐药逆转效果,EC50值分别为高良姜水提物16.37mgL-1和红豆蔻醇提物14.96mgL-1,RT-PCR验证上述两种阳性样品具有明显的抑制MDR1基因表达的作用.以上结果为MDR1基因的转录抑制剂高通量筛选奠定了基础.     

Construction of Multi-ribozyme Expression System and Its Characterization of Cleavage on the MDR1/MRP1 Double Target Substrate in vitro

To improve catalytic activity of ribozyme on its substrate,the multi-ribozyme expression system was designed and constructed from 20 cis-acting hammerhead ribozymes undergoing self-cleavage with 10 trans-acting hammerhead ribozymes inserted altematively regularly and the plasmid of pGEM-MDRI/MRPI used to transcribe the M DRI/MRPI(196/210) substrate containing double target sites was also constructed by DNA recombination.Endonuclease digestion analysis and DNA sequencing indicate all the recombinant plasmids were correct.The cleavage activities were evaluated for the multi-ribozyme expression system on the MDR1/MRP1 substrate in the cell free system.The results demonstrate that the cis-acting hammerhead ribozymes in the multi-ribozyme expression system were able to cleave themselves and the 72 nt of 196Rz and the 71 nt of 210Rz trans-acting hammerhead ribozymes were liberated effectively,and the trans-acting hammerhead ribozymes released were able to act on the MDR1/MRP1 double target RNA substrate and cleave the target RNA at specific sites effectively.The multiribozyme expression system of the [Coat'A196Rz/Coat'B210Rz]5 is more significantly superior to that of the [Coat'A 196Rz/Coat'B210Rz]1 in cleavage of RNA substrate.The fractions cleaved by [Coat'A196Rz/Coat'B210Rz]5 on the MDR1/MRP1 substrate for 8 h at observed temperatures showed no marked difference.The studies of Mg2+ on cleavage efficiency indicate that cleavage reaction is dependent on Mg2+ ions concentration.The plot of Ig(kobs) vs.Igc(Mg2+) displays a linear relationship between 2.5 mmol/L and 20 mmol/L Mg2..It suggests that Mg2+ ions play a crucial role in multi-ribozyme cleavage on the substrate.     

Isobavachalcone as an Active Membrane Perturbing Agent and Inhibitor of ABCB1 Multidrug Transporter

Isobavachalcone (IBC) is an active substance from the medicinal plant Psoralea corylifolia. This prenylated chalcone was reported to possess antioxidative, anti-inflammatory, antibacterial, and anticancer activities. Multidrug resistance (MDR) associated with the over-expression of the transporters of vast substrate specificity such as ABCB1 (P-glycoprotein) belongs to the main causes of cancer chemotherapy failure. The cytotoxic, MDR reversing, and ABCB1-inhibiting potency of isobavachalcone was studied in two cellular models: human colorectal adenocarcinoma HT29 cell line and its resistant counterpart HT29/Dx in which doxorubicin resistance was induced by prolonged drug treatment, and the variant of MDCK cells transfected with the human gene encoding ABCB1. Because MDR modulators are frequently membrane-active substances, the interaction of isobavachalcone with model phosphatidylcholine bilayers was studied by means of differential scanning calorimetry. Molecular modeling was employed to characterize the process of membrane permeation by isobavachalcone. IBC interacted with ABCB1 transporter, being a substrate and/or competitive inhibitor of ABCB1. Moreover, IBC intercalated into model membranes, significantly affecting the parameters of their main phospholipid phase transition. It was concluded that isobavachalcone interfered both with the lipid phase of cellular membrane and with ABCB1 transporter, and for this reason, its activity in MDR cancer cells was presumptively beneficial.     

纳米载体在共负载siRNA及化疗药物对逆转肿瘤多药耐药性方面的应用

近年来,基于小干扰RNA(siRNA)的基因干扰技术从基因水平上调节与肿瘤产生多药耐药性相关的各种蛋白进而逆转化疗多药耐药性方面表现出了巨大的应用潜力.鉴于此,研究者们在RNA干扰与化疗药物的协同抗癌方面做了大量工作.但游离的siRNA在无载体的情况下不易被细胞吸收,而且会被血浆和组织中内源性的核糖核酸酶降解,因此必须...     

New analogs of temporin-LK1 as inhibitors of multidrug-resistant (MDR) bacterial pathogens

The peptide temporin-LK1 (1) was obtained from the skin secretion of frog Limnonectes kuhlii (Ranidae). It is a unique antimicrobial peptide with 17 residues, including four L-phenylalanines and single glycine. Mass spectrometry and Edmand degradation were used for the determination of sequence of amino acids in temporin-LK 1 (1), and confirmed by cDNA cloning. We report here the synthesis and structural studies of temporin-LK1 (1) and its analogs 2–4. Peptides 2–4 were prepared by substitution of achiral glycine residue of temporin-LK1 (1) with D-alanine, L-phenylglycine, and L-naphthylalanine, respectively. Peptides 1–4 were evaluated against multidrug-resistant (MDR) strains of Staphylococcus aureus and Pseudomonas aeruginosa. Analog 2 was found active against all MDR strains of S. aureus and P. aeruginosa at a much lower dose than the clinically used antibiotics.     

Exploring Natural Product Activity and Species Source Candidates for Hunting ABCB1 Transporter Inhibitors: An In Silico Drug Discovery Study

The P-glycoprotein (P-gp/ABCB1) is responsible for a xenobiotic efflux pump that shackles intracellular drug accumulation. Additionally, it is included in the dud of considerable antiviral and anticancer chemotherapies because of the multidrug resistance (MDR) phenomenon. In the search for prospective anticancer drugs that inhibit the ABCB1 transporter, the Natural Product Activity and Species Source (NPASS) database, containing >35,000 molecules, was explored for identifying ABCB1 inhibitors. The performance of AutoDock4.2.6 software to anticipate ABCB1 docking score and pose was first assessed according to available experimental data. The docking scores of the NPASS molecules were predicted against the ABCB1 transporter. Molecular dynamics (MD) simulations were conducted for molecules with docking scores lower than taxol, a reference inhibitor, pursued by molecular mechanics-generalized Born surface area (MM-GBSA) binding energy estimations. On the basis of MM-GBSA calculations, five compounds revealed promising binding affinities as ABCB1 inhibitors with ΔG_binding < −105.0 kcal/mol. The binding affinity and stability of the identified inhibitors were compared to the chemotherapeutic agent. Structural and energetical analyses unveiled great steadiness of the investigated inhibitors within the ABCB1 active site throughout 100 ns MD simulations. Conclusively, these findings point out that NPC104372, NPC475164, NPC2313, NPC197736, and NPC477344 hold guarantees as potential ABCB1 drug candidates and warrant further in vitro/in vivo tests.     

Structural and binding study of modified siRNAs with the Argonaute 2 PAZ domain by NMR spectroscopy

By using high-resolution NMR spectroscopy, the structures of a natural short interfering RNA (siRNA) and of several altritol nucleic acid (ANA)-modified siRNAs were determined. The interaction of modified siRNAs with the PAZ domain of the Argonaute 2 protein of Drosophila melanogaster was also studied. The structures show that the modified siRNA duplexes (ANA/RNA) adopt a geometry very similar to the naturally occurring A-type siRNA duplex. All ribose residues, except for the 3' overhang, show 3'-endo conformation. The six-membered altritol sugar in ANA occurs in a chair conformation with the nucleobase in an axial position. In all siRNA duplexes, two overhanging nucleotides at the 3' end enhance the stability of the first neighboring base pair by a stacking interaction. The first overhanging nucleotide has a rather fixed position, whereas the second overhanging nucleotide shows larger flexibility. NMR binding studies of the PAZ domain with ANA-modified siRNAs demonstrate that modifications in the double-stranded region of the antisense strand have some small effects on the binding affinity as compared with the unmodified siRNA. Modification of the 3' overhang with thymidine (dTdT) residues shows a sixfold increase in the binding affinity compared with the unmodified siRNA (relative binding affinity of 17% compared with dTdT-modified overhang), whereas modification of the 3' overhang with ANA largely decreases the binding affinity.     

Site-specific modification of the 5"-terminal fragment of PGY1/MDR1 gene mRNA by reactive conjugates of antisense oligonucleotides

The site-specific modification of the 5"-terminal fragment of PGY1/MDR1 mRNA by oligodeoxyribonucleotide conjugates bearing residues of bleomycin A₅ (Blm), cobalt(ii) tetracarboxyphthalocyanine (Phcn), 4-[N-(2-chloroethyl)-N-methylamino]benzylamine (RCl), or perfluoroarylazide (Az) was studied. Conjugates of oligonucleotides complementary to the RNA sequences 123—138 and 155—166 selectively modify RNA in the vicinity of these regions. The highest efficacy (up to 50%) was achieved in reactions with alkylating and perfluoroarylazide conjugates of oligonucleotides. Conjugates of perfluoroarylazide with 2"-O-modified oligonucleotides are much more efficient than analogous conjugates with oligodeoxyribonucleotides (extents of RNA modification are 40—50% and 20%, respectively).     

Reversal of Multidrug Resistance by Apolipoprotein A1-Modified Doxorubicin Liposome for Breast Cancer Treatment

Multidrug resistance (MDR) remains a major problem in cancer therapy and is characterized by the overexpression of p-glycoprotein (P-gp) efflux pump, upregulation of anti-apoptotic proteins or downregulation of pro-apoptotic proteins. In this study, an Apolipoprotein A1 (ApoA1)-modified cationic liposome containing a synthetic cationic lipid and cholesterol was developed for the delivery of a small-molecule chemotherapeutic drug, doxorubicin (Dox) to treat MDR tumor. The liposome-modified by ApoA1 was found to promote drug uptake and elicit better therapeutic effects than free Dox and liposome in MCF-7/ADR cells. Further, loading Dox into the present ApoA1-liposome systems enabled a burst release at the tumor location, resulting in enhanced anti-tumor effects and reduced off-target effects. More importantly, ApoA1-lip/Dox caused fewer adverse effects on cardiac function and other organs in 4T1 subcutaneous xenograft models. These features indicate that the designed liposomes represent a promising strategy for the reversal of MDR in cancer treatment.     

Downregulation of Orai1 expression in the airway alleviates murine allergic rhinitis

Orai1 is the key subunit of the Ca(2+)-release-activated Ca(2+) channel. Our previous report has demonstrated that Orai1 expression in the airway was upregulated in the ovalbumin (OVA)-induced allergic rhinitis (AR) mouse models. To observe whether inhibition of Orai1 expression in the airway could suppress symptoms in a murine model of AR and to assess the impacts of this inhibition on the responses of local and systemic immunocytes, we administered recombinant lentivirus vectors that encoded shRNA against ORAI1 (lenti-ORAI1) into the nostrils of OVA-sensitized mice before the challenges, and analyzed its effect on allergic responses, as compared with the unsensitized mice and untreated AR mice. Administration of lenti-ORAI1 into the nasal cavity successfully infected cells in the epithelial layer of the nasal mucosa, and significantly decreased the frequencies of sneezing and nasal rubbing of the mice. Protein levels of leukotriene C4, OVA-specific IgE, and IL-4 in the nasal lavage fluid and serum and eosinophil cation protein in the serum were also significantly reduced by lenti-ORAI1, as were the mRNA levels of these factors in the nasal mucosa and spleen. These data suggested that administration of lenti-ORAI1 into the nasal cavity effectively decreased Orai1 expression in the nasal mucosa, alleviated AR symptoms, and partially inhibited the hyperresponsiveness of the local and systemic immune cells including T cells, B cells, mast cells and eosinophils that are involved in the pathogenesis of AR.     

Prediction and analysis of binding affinities for chemically diverse HIV-1 PR inhibitors by the modified SAFE_p approach

One of the biggest challenges in the "in silico" screening of enzyme ligands is to have a protocol that could predict the ligand binding free energies. In our group we have developed a very simple screening function (referred to as solvent accessibility free energy of binding predictor, SAFE_p) which we have applied previously to the study of peptidic HIV-1 protease (HIV-1 PR) inhibitors and later to cyclic urea type HIV-1 PR inhibitors. In this work, we have extended the SAFE_p protocol to a chemically diverse set of HIV-1 PR inhibitors with binding constants that differ by several orders of magnitude. The resulting function is able to reproduce the ranking and in many cases the value of the inhibitor binding affinities for the HIV-1 PR, with accuracy comparable with that of costlier protocols. We also demonstrate that the binding pocket SAFE_p analysis can contribute to the understanding of the physical forces that participate in ligand binding. The analysis tools afforded by our protocol have allowed us to identify an induced fit phenomena mediated by the inhibitor and have demonstrated that larger fragments do not necessarily contribute the most to the binding free energy, an outcome partially brought about by the substantial role the desolvation penalty plays in the energetics of binding. Finally, we have revisited the effect of the Asp dyad protonation state on the predicted binding affinities.     

Quantitative 1H NMR analysis of reacted silanol groups in silica nanoparticles chemically modified with monochlorosilanes

Quantitative analysis of reacted silanol groups in silica nanoparticles modified chemically with monochlorosilanes was performed by ¹H NMR after treatment with cesium fluoride. Silica nanoparticles were modified chemically by the reaction between the silanol groups and monochlorosilanes, and the structure of the organic moiety anchored onto the silica surface was confirmed with solid‐state ¹³C NMR. As monochlorosilanes react with silanol groups at 1:1 ratio unlike di‐ or trichlorosilanes, the number of the silanes introduced into silica nanoparticles equals that of reacted silanol groups. Organically modified silica nanoparticles were dissolved using cesium fluoride, and the amount of the soluble organic compounds originated from the introduced silanes was determined by a ¹H NMR internal standard method using pyrene as the reference. Those values determined by ¹H NMR were in good agreement with those determined by elemental analysis. Thus, the number of reacted silanol groups per one particle was calculated on the basis of the results obtained by the ¹H NMR method, and the values were highly dependent on the steric structure of the introduced silanes. Copyright © 2010 John Wiley & Sons, Ltd.     

单分子力谱研究活细胞上多药耐药相关蛋白1的表达

采用原子力显微镜的单分子力谱(SMFM)技术研究了多药耐药相关蛋白1(MRP1)与其抗体间的相互作用, 并考察了人舌癌细胞系TCA8113经高剂量平阳霉素(BLM)反复间歇诱导前后细胞表面MRP1的表达差异. 实验结果表明, MRP1与其抗体之间存在特异性相互作用力, 当针尖运动速率为2.5 μm/s时, 作用力大小约为(182±35) pN; 而且药物诱导后MRP1在人舌癌细胞上的表达明显增强. 本工作为了解活细胞水平上MRP1的表达提供了新方法, 有助于肿瘤细胞多药耐药性(MDR)的研究.     

Effects of Two Natural Bisbenzylisoquinolines,Curine and Guattegaumerine,Extracted from Isolona hexaloba on Rhodamine Efflux by Abcb1b from Rat Glycocholic-Acid-Resistant Hepatocarcinoma Cells

To develop new therapeutic molecules, it is essential to understand the biological effects and targets of clinically relevant compounds. In this article, we describe the extraction and characterization of two alkaloids from the roots of Isolona hexaloba—curine and guattegaumerine. The effect of these alkaloids on the multidrug efflux pump ABCB1 (MDR1/P-Glycoprotein) and their antiproliferative properties were studied. Compared to verapamil, a widely used inhibitor of P-gp, curine and guattegaumerine were found to be weak inhibitors of MDR1/P-Glycoprotein. The highest inhibition of efflux produced by verapamil disappeared in the presence of curine or guattegaumerine as competitors, and the most pronounced effect was achieved with curine. Altogether, this work has provided new insights into the biological effects of these alkaloids on the rat Mdr1b P-gp efflux mechanism and would be beneficial in the design of potent P-gp inhibitors.