Discovery of Novel Symmetrical 1,4-Dihydropyridines as Inhibitors of Multidrug-Resistant Protein (MRP4) Efflux Pump for Anticancer Therapy

Despite the development of targeted therapies in cancer, the problem of multidrug resistance (MDR) is still unsolved. Most patients with metastatic cancer die from MDR. Transmembrane efflux pumps as the main cause of MDR have been addressed by developed inhibitors, but early inhibitors of the most prominent and longest known efflux pump P-glycoprotein (P-gp) were disappointing. Those inhibitors have been used without knowledge about the expression of P-gp by the treated tumor. Therefore the use of inhibitors of transmembrane efflux pumps in clinical settings is reconsidered as a promising strategy in the case of the respective efflux pump expression. We discovered novel symmetric inhibitors of the symmetric efflux pump MRP4 encoded by the ABCC4 gene. MRP4 is involved in many kinds of cancer with resistance to anticancer drugs. All compounds showed better activities than the best known MRP4 inhibitor MK571 in an MRP4-overexpressing cell line assay, and the activities could be related to the various substitution patterns of aromatic residues within the symmetric molecular framework. One of the best compounds was demonstrated to overcome the MRP4-mediated resistance in the cell line model to restore the anticancer drug sensitivity as a proof of concept.     

具有肿瘤多药耐药逆转作用的金雀异黄素衍生物的合成及生物活性研究

细胞膜P-糖蛋白(P-gp)介导的药物外流是肿瘤多药耐药(MDR)产生的重要机制,异黄酮类化合物可以通过抑制P-gp活性发挥MDR逆转作用.通过对P-gp抑制剂进行结构分析,以金雀异黄素为母体,在其7位、8位及4'位分别引进碱性边链,设计、合成了20个金雀异黄素衍生物(其中16个未见文献报道),并检测了其多药耐药逆转活性.结果表明,大多数目标化合物对人白血病耐药细胞株K562/A02具有不同程度的耐药逆转作用.其中目标化合物8a,8b,8d,8e逆转作用较强,逆转倍数分别为8.97,6.36,5.19和5.82.     

Ethyl lucidenates A reverses P-glycoprotein mediated vincristine resistance in K562/A02 cells

Multidrug resistance is a major unresolved obstacle to successful cancer chemotherapy. It is often associated with an elevated efflux of a variety of anticancer drugs by ATP-binding cassette transporters including P-glycoprotein, BCRP and MRP1. In this study, the reversal effect of Ethyl lucidenates A on K562/A02 cells was investigated. At concentrations of 10 μM, Ethyl lucidenates A could reverse the resistance of K562/A02 to vincristine up to 7.59 folds. Mechanistically, Ethyl lucidenates A could increase the intracellular accumulation of vincristine in K562/A02 cells through inhibiting the P-glycoprotein mediated drug-transport activity by rhodamine accumulation assay and cell cycle analysis. Further mechanistic investigation found that Ethyl lucidenates A did not alter P-glycoprotein expression. In conclusion, Ethyl lucidenates A could reverse the multidrug resistance of K562/A02 cells via its influence on P-glycoprotein drug-transport activity and thus, be a potential multidrug resistance reversal agent.     

In situ monitoring of functional activity of extracellular matrix stiffness-dependent multidrug resistance protein 1 using scanning electrochemical microscopy

Extracellular matrix (ECM) stiffness affects the drug resistance behavior of cancer cells, while multidrug resistance protein 1 (MRP1) on the cell membrane confers treatment resistance via actively transporting drugs out of cancer cells. However, the relationship between ECM stiffness and MRP1 functional activity in cancer cells remains elusive, mainly due to the technical challenge of in situ monitoring. Herein, we engineered in vitro cancer cell models using breast cancer cells (MCF-7 and MDA-MB-231 cells) as the reprehensive cells on polyacrylamide (PA) gels with three stiffness, mimicking different developmental stages of cancer. We in situ characterized the functional activity of MRP1 and investigated the effect of ECM stiffness on MRP1 of cancer cells before and after vincristine treatment using scanning electrochemical microscopy (SECM) with ferrocenecarboxylic acid (FcCOOH) as the redox mediator and endogenous glutathione (GSH) as the indicator. The SECM results show that the functional activity of MRP1 is enhanced with increasing ECM stiffness, and the MRP1-mediated vincristine efflux activity of MCF-7 cells is more affected by ECM stiffness than that of MDA-MB-231 cells. This work, for the first time, applied SECM to in situ and quantitatively monitor the functional activity of MRP1 in cancer cells in different tumor mechanical microenvironments, which could help to elucidate the mechanism of matrix stiffness-dependent drug resistance behavior in cancer cells.

SECM using FcCOOH as the redox mediator and endogenous GSH as the indicator was employed to investigate the effect of extracellular matrix stiffness on the functional activity of MRP1 in cancer cells in situ.     

Doxorubicin Encapsulated in TPGS-Modified 2D-Nanodisks Overcomes Multidrug Resistance

Multidrug resistance (MDR) is regarded as a main obstacle for effective chemotherapy, and P-glycoprotein (P-gp)-mediated drug efflux has been demonstrated to be the key factor responsible for MDR. In this study, a novel pH-responsive hybrid drug delivery system was developed by conjugating d -α-tocopheryl polyethylene glycol 1000 succinate (TPGS), a kind of P-gp inhibitor, on the surface of laponite nanodisks to overcome MDR. The prepared LM-TPGS display excellent colloidal stability, a high encapsulation efficiency of doxorubicin (DOX), and a pH-responsive drug release profile. In vitro experiments verified that LM-TPGS/DOX could exhibit significantly enhanced therapeutic efficacy in treating DOX-resistant breast cancer cells (MCF-7/ADR) through inhibiting the activity of P-gp-mediated drug efflux and effectively accumulating DOX within cancer cells. In vivo results revealed that LM-TPGS/DOX outstandingly suppressed MCF-7/ADR tumors with low side effects. Therefore, the high drug payload, enhanced inhibition efficacy to drug-resistant cells, and low side effects make the LM-TPGS/DOX a promising nanoplatform to reverse MDR for effective chemotherapy.     

New cembranolide analogues from the formosan soft coral Sinularia flexibilis and their cytotoxicity

Using a bioactivity-guided fractionation procedure, five cembranolides, 11-epi-sinulariolide acetate (1), 11-dehydrosinulariolide (2), sinulariolide (3), dihydrosinularin (4), and 3,4:8,11-bisepoxy-7-acetoxycembra-15(17)-en-1,12-olide (5), along with two nucleosides, 2'-deoxyadenosine and thymidine, were isolated from the Formosan soft coral Sinularia flexibilis. Moreover, 7,8-epoxy-11-epi-sinulariolide acetate (1a), 11-sinulariolide acetate (3a), dihydrosinulariolide (3b), 3,4:8,11-bisepoxy-7-hydroxycembra-15(17)-en-1,12-olide (3c), 11-acetoxyl-15(17)-dihydrosinulariolide (3d), 7,8-epoxy-11-sinulariolide acetate (3e), and 3,4:8,11-bisepoxy-7-hydroxycembra-15(17)-dihydro-1,12-olide (3f) were derived from compounds 1 and 3, respectively. These structures were deduced on the basis of physical and chemical evidence. Among them, 1a, 3d, 3e, and 3f are new cembranolide analogues. The structure of compound 1 was further confirmed by X-ray analysis. In addition, the isolated cembranolides and the analogues under went a cytotoxicity assay, and the structure-activity relationship (SAR) of these compounds was studied.     

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.     

7-硫代鸟嘌呤互变异构体的密度泛函理论计算

在密度泛函B3LYP/6-311G^**水平下,对14种气相和水相中可能存在的7-硫代鸟嘌呤异构体进行了几何构型全自由度优化,并计算出它们的总能量、焓、熵、吉布斯自由能.Onsager反应场溶剂模型用于水相的计算.计算结果表明,7-硫代鸟嘌呤在气相中和水相中主要以硫酮形式存在.在气相中,硫酮-N(6)(H)要比硫酮-N(4)(H)更稳定,而在水相中,则硫酮-N(4)(H)要比硫酮-N(6)(H)更稳定.计算结果同已有实验结果一致.7-硫代鸟嘌呤互变异构的熵效应小,对互变异构平衡没有显著的影响,而焓变对互变异构产生了主要的影响.较详细地讨论了水溶剂化作用对异构体的能量、几何结构、电荷分布和偶极矩的影响.溶剂化自由能与异构体的偶极矩存在相关性.     

Investigation of the Uptake and Transport of Two Novel Camptothecin Derivatives in Caco-2 Cell Monolayers

Irinotecan and Topotecan are two Camptothecin derivatives (CPTs) whose resistance is associated with the high expression of breast cancer resistance protein (BCRP) and P-glycoprotein (P-gp). To reverse this resistance, two novel CPTs, FL77-28 (7-(3-Fluoro-4-methylphenyl)-10,11-methylenedioxy-20(S)-CPT) and FL77-29 (7-(4-Fluoro-3-methylphenyl)-10,11-methylenedioxy-20(S)-CPT), were synthesized by our group. In this study, the anti-tumor activities of FL77-28, FL77-29, and their parent, FL118 (10,11-methylenedioxy-20(S)-CPT), were evaluated and the results showed that FL77-28 and FL77-29 had stronger anti-tumor activities than FL118. The transport and uptake of FL118, FL77-28, and FL77-29 were investigated in Caco-2 cells for the preliminary prediction of intestinal absorption. The apparent permeability coefficient from apical to basolateral (P_{app AP-BL}) values of FL77-28 and FL77-29 were (2.32 ± 0.04) × 10⁻⁶ cm/s and (2.48 ± 0.18) × 10⁻⁶ cm/s, respectively, suggesting that the compounds had moderate absorption. Since the transport property of FL77-28 was passive diffusion and the efflux ratio (ER) was less than 2, two chemical inhibitors were added to further confirm the involvement of efflux proteins. The results showed that FL77-28 was not a substrate of P-gp or BCRP, but FL77-29 was mediated by P-gp. In conclusion, FL77-28 might be a promising candidate to overcome drug resistance induced by multiple efflux proteins.     

A 3D QSAR pharmacophore model and quantum chemical structure--activity analysis of chloroquine(CQ)-resistance reversal

Using CATALYST, a three-dimensional QSAR pharmacophore model for chloroquine(CQ)-resistance reversal was developed from a training set of 17 compounds. These included imipramine (1), desipramine (2), and 15 of their analogues (3-17), some of which fully reversed CQ-resistance, while others were without effect. The generated pharmacophore model indicates that two aromatic hydrophobic interaction sites on the tricyclic ring and a hydrogen bond acceptor (lipid) site at the side chain, preferably on a nitrogen atom, are necessary for potent activity. Stereoelectronic properties calculated by using AM1 semiempirical calculations were consistent with the model, particularly the electrostatic potential profiles characterized by a localized negative potential region by the side chain nitrogen atom and a large region covering the aromatic ring. The calculated data further revealed that aminoalkyl substitution at the N5-position of the heterocycle and a secondary or tertiary aliphatic aminoalkyl nitrogen atom with a two or three carbon bridge to the heteroaromatic nitrogen (N5) are required for potent "resistance reversal activity". Lowest energy conformers for 1-17 were determined and optimized to afford stereoelectronic properties such as molecular orbital energies, electrostatic potentials, atomic charges, proton affinities, octanol-water partition coefficients (log P), and structural parameters. For 1-17, fairly good correlation exists between resistance reversal activity and intrinsic basicity of the nitrogen atom at the tricyclic ring system, frontier orbital energies, and lipophilicity. Significantly, nine out of 11 of a group of structurally diverse CQ-resistance reversal agents mapped very well on the 3D QSAR pharmacophore model.     

Phenolic Constituents from Platycodon grandiflorum Root and Their Anti-Inflammatory Activity

Six lignols (1–6), including two new compounds (+)-(7R,8R)-palmitoyl alatusol D (1) and (+)-(7R,8R)-linoleyl alatusol D (2), along with four phenolics (7–10), a neolignan (11), three alkyl aryl ether-type lignans (12–14), two furofuran-type lignans (15–16), three benzofuran-type lignans (17–19), a tetrahydrofuran-type lignan (20), and a dibenzylbutane-type lignan (21) were isolated from the ethyl acetate-soluble fraction of the methanol extract of Platycodon grandiflorum (Jacq.) A. DC. root. The chemical structures of the obtained compounds were elucidated via high-resolution mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy analyses. The obtained spectroscopic data agreed well with literature. Among the isolated compounds, eighteen (1–7 and 11–21) were isolated from P. grandiflorum and the Campanulaceae family for the first time. This is the first report on lignol and lignan components of P. grandiflorum. The anti-inflammatory effects of the isolated compounds were examined in terms of their ability to inhibit the production of pro-inflammatory cytokines IL-6, IL-12 p40, and TNF-α in lipopolysaccharide-stimulated murine RAW264.7 macrophage cells. Nine compounds (4–6, 12, and 15–19) exhibited inhibitory effects on IL-12 p40 production, eleven compounds (1–6, 12, 15–17, and 19) exhibited inhibitory activity on IL-6 production, and eleven compounds (1–6 and 15–19) exhibited inhibitory effects against TNF-α. These results warrant further investigation into the potential anti-inflammatory activity and general benefits of the phenolic constituents of P. grandiflorum root.     

新型哌啶-查尔酮类衍生物的设计、合成及抗宫颈癌和逆转顺铂耐药活性

本文采用活性亚结构拼接原理,设计并合成了15个新型含哌啶的查尔酮类衍生物,利用¹H NMR、¹³C NMR和HR-MS对结构进行表征,并初步评价了其抗宫颈癌和抗顺铂耐药宫颈癌活性作用。结果表明,化合物6g具有一定的抗肿瘤活性和逆转顺铂耐药作用;并采用Elisa法、联合顺铂用药、Western Blot和分子对接对化合物6g与VEGFR-2和P-gp靶点进行了初步的研究。本研究为基于VEGFR-2和P-gp双靶点新型分子靶向查尔酮类衍生物的设计提供了一条思路。     

A Dual-Column HPLC Method for the Simultaneous Measurement of 6-Thioguanine and Adenine in RNA or DNA

Abstract

A sensitive method for measuring 6-thioguanine incorporation into DNA and RNA utilizing a dual column system is presented. The measurement of the 6-thioguanine deoxyribo-or ribonucleosides and deoxyadenosine or adenosine is made simultaneously, thereby allowing for direct calculation of the incorporation per nucleic acid base. The separation utilizes a strong anion-exchange column connected in series with an octadecylsilane column. Prior to high pressure liquid chromatography, the sample is partially purified and oxidized with potassium permanganate. Following a 10-min delay, a 10-min linear gradient from 2% to 20% methanol in 30 mM NH₄H₂PO₄, pH 3.7, is employed. Detection of eluting material is by fluorescence and by UV absorbance at 254 nm. Recovery of the 6-thioguanine nucleosides was determined using [8-¹⁴C]-6-thioguanine. The sensitivity of the method for the oxidized 6-thioguanine compounds is approximately 1 pmole (fluorescence) whereas that for the adenine nucleosides (UV absorbance) is about 100 pmoles. This sensitivity is adequate to determine the incorporation in less than 10⁶ (about 1 mg) Chinese hamster ovary cells exposed to a cytotoxic concentration of 6-thioguanine.     

The synthesis and evaluation of a solution phase indexed combinatorial library of non-natural polyenes for reversal of P-glycoprotein mediated multidrug resistance

A combinatorial library of polyenes, based on (-)-stipiamide, has been constructed and evaluated for the discovery of new multidrug resistance reversal agents. A palladium coupling was used to react each individual vinyl iodide with a mixture of the seven acetylenes at near 1:1 stoichiometry. The coupling was also used to react each individual acetylene with the mixture of six vinyl iodides to create 13 pools indexed in two dimensions for a total of 42 compounds. Individual compounds were detected at equimolar concentration. The vinyl iodides, made initially using a crotylborane addition to generate the anti1,2-hydroxylmethyl products, were now made using a more efficient norephedrine propionate boron enolate aldol reaction. The indexed approach, ideally suited for cellular assays that involve membrane-bound targets, allowed for the rapid identification of reversal agents using assays with drug-resistant human breast cancer MCF7-adrR cells. Intersections of potent pools identified new compounds with promising activity. Aryl dimension pools showed R = ph and naphthyl as the most potent. The acetylene dimension had R' = phenylalaninol and alaninol as the most potent. Isolated individual compounds, both active and nonpotent, were assayed to confirm the library results. The most potent new compound was 4ek (R = naphthyl, R' = phenylaninol) at 1.45 microM. Other nonnatural individual naphthyl-amide compounds showed potent MDR reversal including the morpholino-amide 4ej (1.69 microM). Synergistic activities attributed to the two ends of the molecule were also identified. Direct interaction with Pgp was established by ATPase and photoaffinity displacement assays. The results indicate that both ends of the polyene reversal agent are involved in Pgp interaction and can be further modified for increased potency.     

Synthesis of Pyridotriazolopyrimidines as Antitumor Agents

2‐Hydrazino‐5,7‐di‐p‐tolylpyrido[2,3‐d ]pyrimidin‐4(3H )‐one ( 4 ) was prepared and condensed with different aldehydes 5a , 5b , 5c , 5d , 5e , 5f , 5g to give the corresponding hydrazone derivatives 6a , 6b , 6c , 6d , 6e , 6f , 6g . Oxidative cyclization of the latter compounds 6a , 6b , 6c , 6d , 6e , 6f , 6g gave the corresponding pyrido[2,3‐d ][1,2,4]triazolo[4,3‐a ]pyrimidin‐5(1H )‐ones 7a , 7b , 7c , 7d , 7e , 7f , 7g . Furthermore, compound 4 reacted with benzoyl chloride, triethyl orthoformate, acetyl chloride, ethyl chloroformate, and carbon disulphide in alcoholic KOH solution to afford the corresponding pyrido[2,3‐d ][1,2,4]triazolo[4,3‐a ]pyrimidinones ( 7a , 8 , 9 , 10 , 11 ). The reaction of thione 3 or its 2‐methylthio derivative 16 with hydrazonoyl halides 12a , 12b , 12c , 12d , 12e , 12f , 12g , 12h , 12i , 12j , 12k , 12l , 12m yielded the corresponding pyrido[2,3‐d ][1,2,4]triazolo[4,3‐a ]pyrimidinones 15a , 15b , 15c , 15d , 15e , 15f , 15g , 15h , 15i , 15j , 15k , 15l , 15m . The structures of all the products were confirmed by elemental and spectral analyses (¹H NMR, ¹³C NMR, IR, and MS). In addition, the anticancer activity of 20 pyridotriazolopyrimidinones against two cancer cell lines namely MCF‐7 and HepG2 was evaluated, and the results revealed that compounds 7d and 9 have promising activity , compared with doxorubicin, which used as standard reference drug.     

Lamellarins as Inhibitors of P‐Glycoprotein‐Mediated Multidrug Resistance in a Human Colon Cancer Cell Line

Chemical analysis of a Didemnum sp. (CMB‐01656) collected during scientific Scuba operations off Wasp Island, New South Wales, yielded five new lamellarins A1 ( 1 ), A2 ( 2 ), A3 ( 3 ), A4 ( 4 ) and A5 ( 5 ) and eight known lamellarins C ( 6 ), E ( 7 ), K ( 8 ), M ( 9 ), S ( 10 ), T ( 11 ), X ( 12 ) and χ ( 13 ). Analysis of a second Didemnum sp. (CMB‐02127) collected during scientific trawling operations along the Northern Rottnest Shelf, Western Australia, yielded the new lamellarin A6 ( 14 ) and two known lamellarins G ( 15 ) and Z ( 16 ). Structures were assigned to 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 on the basis of detailed spectroscopic analysis with comparison to literature data and authentic samples. Access to this unique library of natural lamellarins ( 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 ) provided a rare opportunity for structure–activity relationship (SAR) investigations, probing interactions between lamellarins and the ABC transporter efflux pump P‐glycoprotein (P‐gp) with a view to reversing multidrug resistance in a human colon cancer cell line (SW620 Ad300). These SAR studies, which were expanded to include the permethylated lamellarin derivative ( 17 ) and a series of lamellarin‐inspired synthetic coumarins ( 19 , 20 , 21 , 22 , 23 , 24 ) and isoquinolines ( 25 , 26 ), successfully revealed 17 as a promising new non‐cytotoxic P‐gp inhibitor pharmacophore.     

Triazolopyridines. Part 8.1 Nucleophilic substitution reactions of 5-bromo[1,2,3]triazolo[5,1-a]isoquinoline and 7-bromo[1,2,3]-triazolo[1,5-a]pyridine

Nucleophilic substitution of 5-bromotriazoloisoquinoline (3) and of 7-bromo-3-methyltriazolopyridine (6) proceeds readily to give a range of 5-substituted triazoloisoquinolines (4a)-(4e), and of 7-substituted triazolopyridines (7a)-(7h) respectively. Triazoloisoquinolines have been converted into 1,3-disubstituted isoquinolines (11)-(13), (15), and (16), and triazolopyridines into 2,6-disubstituted pyridines (17)-(19). Of secondary amine nucleophiles, only piperidine reacted with 7-bromo-3-methyltriazolopyridine (6) to give the 7-substituted derivative (7g). A second product in this reaction was a 2,6-disubstituted pyridine (8); the similar compounds (20)-(24) were the only products when morpholine or N-acetylpiperazine were used. The reaction between 7-bromotriazolopyridine (9) and piperidine or morpholine gave in high yield the 2,6-disubstituted pyridines (25) and (26).     

The reaction of active and stabilized phosphonium ylides with α,β-unsaturated carbonyl compounds [1]

The active ketenylidene-(2a) or thioketenylidenetriphenylphosphoranes (2b) react with 2-benzylidene-1,3-indandione (1), 5-benzylidenebarbituric acid (11), and 4-benzylidene-1,2-diphenyl-3,5-pyrazolidinedione (16) to give the corresponding pyranones and thioxopyranones (3a,b, 12a,b) and (17a,b), respectively. On the other hand, compounds 1 and 11 can be converted by reaction with the stabilized alkylidenephosphoranes 4a–e into the phosphoranylidenes 6a–e and 13a–e. Moreover, the oxaphosphinins 8 or 14 and the oxazaphosphinins 10 or 15 were obtained when compounds 1 and 11 were allowed to react with the phosphorane 7 and the iminophosphorane 9, respectively. Some of these new organophosphorus compounds are found to have insecticidal and molluscicidal properties against cotton leafworm Spodoptera littoralis larvae and Biomphalaria alexandrina snails. © 1997 John Wiley, & Sons, Inc.     

GENETIC EFFECTS OF UV-B: MUTAGENICITY OF 308 nm LIGHT IN CHINESE HAMSTER V79 CELLS

Abstract —Chinese hamster V79 cells were irradiated with 254 nm (UV-C) and 308 nm (UV-B) light, emitted by a germicidal lamp and an excimer laser, respectively. Induction of mutations at two distinct genetic loci was measured by selecting colonies resistant to 6-thioguanine or to ouabain. Unlike 6-thioguanine resistance which can be presumed to be due to many different types of genetic damage, mutation to ouabain resistance seems to result from base-pair substitution events only. Much higher doses of 308 than of 254 nm radiation are required to induce equivalent numbers of mutants. However, induction of cell inactivation and 6-thioguanine resistant mutations with the two UV sources appears to be correlated, suggesting that a common mechanism, perhaps involving the induction of pyrimidine-containing dimers, is involved. The frequency of ouabain resistant mutants per lethal event is on the other hand much higher after irradiation with the 308 nm light. This latter finding further defines a part of the UV-B spectral region which seems to induce a unique kind of DNA damage which specifically results in base-pair substitution events. Action spectra studies therefore appear necessary in the definition of the mutagenic effects of UV-B radiations in mammalian cells.     

Rapid determination of multidrug resistance-associated protein in cancer cells by capillary electrophoresis immunoassay

The adenosine triphosphate (ATP) binding-cassette (ABC) transporters are a superfamily of cellular proteins that have been partly implicated as a cause of multidrug resistance (MDR) in cancer cells. The ABC superfamily consists of P-glycoprotein, multidrug resistance-associated proteins (MRP) and breast cancer-related proteins, of which MRP is of particular interest because of its ability to efflux a broader range of substrates. Since MRP1 is the most prominent member of the MRP family, a simple technique is needed for its quantification. We developed a simple, fast (total analysis time of 3h) capillary electrophoresis immunoassay (CEIA) for the quantification of MRP1 in cancer cells. MRP1 antibody was labeled with fluorescein isothiocyanate. The labeled antibody was incubated with the cell lysate for a fixed interval (1h), after which the cell lysate mixture was directly injected into the capillary to separate the complex of MRP1 and its antibody from free antibody. The noncompetitive CEIA method had a limit of detection of 0.2 nM and a good linear range (1.7-14.9×10(4) cells), and was fairly reproducible (RSD<10%). The results showed that two cell lines, A549 and RDES, expressed MRP1 in the absence of doxorubicin (DOX), with A549 registering a higher expression. Compared to DOX-free cancer cells, there was an acceleration of MRP1 expression during the 12h-exposure to DOX, after which the level of expression remained nearly constant as the intracellular accumulation of DOX decreased. The results obtained in this work indicate that the developed CEIA method is useful for relative quantification of MRPs in cancer cells.