Relation between the ability of some compounds to modulate the MRP1-mediated efflux of glutathione and to inhibit the MRPl-mediated efflux of daunorubicin

Much effort has been recently directed to identify the transport-modulating agents in order to overcome the P-gp- and MRP1-mediated drug resistance. Contrary to what is observed for P-gp, very few compounds have been shown to reverse multi-drug resistance (MDR) mediated by MRP1. On the other hand, despite of critical role of GSH in transporting the MRP1 substrates, not much is known about GSH interactions with MRP1. In this work, three compounds that were shown to inhibit the MRP1-mediated efflux of daunorubicin (DNR) have been studied. Depending on their nature the selected compounds have different effects, e.g. at 40 microM, verapamil inhibits 50% of DNR efflux whereas GSH efflux is increased about two-fold. PAK-104P has shown the same effect, i.e. the inhibition of the MRP1-mediated efflux of DNR is accompanied by a stimulation of GSH efflux. However, the PAK-104P concentration required to obtain the same effect is about 40 times smaller that in the case of verapamil. MK571 has been shown to inhibit the efflux of both DNR and GSH. Based on these observations and those reported earlier, a working model is proposed.     

Kinetic analysis of fluorescein and dihydrofluorescein effluxes in tumour cells expressing the multidrug resistance protein,MRP1

Multidrug resistance (MDR) in tumour cells is often caused by the overexpression of two transporters the P-glycoprotein (P-gp) and the multidrug resistance-associated protein (MRP1) which actively pump out multiple chemically unrelated substrates across the plasma membrane. A clear distinction in the mechanism of translocation of substrates by MRP1 or P-gp is indicated by the finding that, in most of cases, the MRP1-mediated transport of substrates is inhibited by depletion of intracellular glutathione (GSH), which has no effect on their P-gp-mediated transport. The aim of the present study was to quantitatively characterise the transport of anionic compounds dihydrofluorescein and fluorescein (FLU). We took advantage of the intrinsic fluorescence of FLU and performed a flow cytometric analysis of dye accumulation in the wild-type drug sensitive GLC4 that do not express MRP1 and its MDR subline which display high level of MRP1. The measurements were made in real time using intact cells. The kinetics parameters, k(a)=V(M)/K(m), which is a measure of the efficiency of the transporter-mediated efflux of a substrate, was very similar for the two FLU analogues. They were highly comparable with values for k(a) of other negatively charged substrates, such as GSH and calcein. The active efflux of both FLU derivatives was inhibited by GSH depletion.     

4-甲基-香豆素及4-甲基喹啉-2(1H)-酮的7位羟基、巯基与炔丙基卤代物的不同反应性

目的合成具有抗HIV活性的三环杂环化合物的关键中间体.方法 7-羟基-4-甲基-香豆素、7-羟基-4-甲基喹啉-2(1H)-酮、7-巯基-4-甲基-香豆素分别与3-氯-3-甲基-1-丁炔、3-溴丙炔反应得到相应产物,其结构经波谱确证.结果 4-甲基-香豆素的7位羟基发生正常的双分子亲核取代反应(SN2),得到炔丙基醚产物4、7和10,进一步热环合得到三环杂环化合物5、8和11;7-巯基-4-甲基-香豆素、7-巯基-4-甲基喹啉-2(1H)-酮与3-氯-3-甲基-1-丁炔反应分别得丙二烯醚双分子亲核取代反应(SN2′)产物聚集双键硫醚化合物12和14,且不能进一步热环合成三环杂环.结论 4-甲基-香豆素及4-甲基喹啉-2(1H)-酮的7位羟基、巯基与炔丙基卤代物表现出不同的反应性.     

1-Ethyl-4-(isopropylidenehydrazino)-1H-pyrazolo-(3,4-b)-pyridine-5-carboxylic acid, ethyl ester, hydrochloride (SQ 20009)--a potent new inhibitor of cyclic 3',5'-nucleotide phosphodiesterases

The properties of SQ 20009 [1-ethyl-4-(isopropylidenehydrazino)-1H-pyrazolo-(3,4-b)-pyridine-5-carboxylic acid, ethyl ester, HCl] as a cyclic nucleotide phosphodiesterase inhibitor have been investigated. The phosphodiesterase preparations used in this study were ammonium sulfate-fractionated supernatants of homogenates of rat brain, rabbit brain, rat adrenal, rat lipocyte and cat heart; commercially available beef heart phosphodiesterase was also studied. The concentrations of SQ 200009 required to inhibit these phosphodiesterase activities 50 per cent were 2·0, 4·8, 20, 21, 27 and 60 μM, respectively, using 1·6 × 10⁻⁷ M cyclic AMP as substrate. SQ 20006 (the parent of SQ 20009 lacking the 4-isopropylidene moiety), theophylline and caffeine were also tested against all six enzyme preparations. Whereas SQ 20009 was more potent than SQ 20006 using the phosphodiesterase prepared from rat adrenal, the potencies were reversed when the lipocyte enzyme was used. SQ 20009 was approximately 60 and 75 times as potent an inhibitor of rat brain cyclic AMP phosphodiesterase as were theophylline and caffeine respectively. The kinetic properties of the phosphodiesterases prepared from rat brain, cat heart and beef heart were also investigated. Using the rat brain enzyme, two K_m values for cyclic AMP, 4·0 × 10⁻⁶ and 1·2× 10⁻⁴ M and a single K_m, 2·0 × 10⁻⁵ M, for cyclic GMP were confirmed. The K_i of SQ 20009 against the low K_m cyclic AMP phosphodiesterase was 2·0 × 10⁻⁶ M and that for cyclic GMP hydrolysis was 2·4 × 10⁻⁵ M. The inhibition by SQ 20009 of the hydrolysis of both cyclic nucleotides by both the rat brain and beef heart phosphodiesterases was competitive. The cat heart cyclic nucleotide phosphodiesterase was inhibited non-competitively by SQ 20009; the K_i for cyclic AMP hydrolysis was 6·4 × 10⁻⁵ M, and the K_i for cyclic GMP hydrolysis was 3·0 × 10⁻⁵ M. The inhibition by SQ 20009 of cyclic AMP hydrolysis by both the rat brain and cat heart preparations was reversible.     

MRP1 mutated in the L0 region transports SN-38 but not leukotriene C4 or estradiol-17 (beta-D-glucuronate)

Multidrug resistance protein 1 (MRP1) is an ATP-binding cassette transporter that confers multidrug resistance on tumor cells. Much convincing evidence has accumulated that MRP1 transports most substances in a GSH-dependent manner. On the other hand, several reports have revealed that MRP1 can transport some substrates independently of GSH; however, the importance of GSH-independent transport activity is not well established and the mechanistic differences between GSH-dependent and -independent transport by MRP1 are unclear. We previously demonstrated that the amino acids W261 and K267 in the L0 region of MRP1 were important for leukotriene C4 (LTC4) transport activity of MRP1 and for GSH-dependent photolabeling of MRP1 with azidophenyl agosterol-A (azidoAG-A). In this paper, we further tested the effect of W222L, W223L and R230A mutations in MRP1, designated dmL0MRP1, on MRP1 transport activity. SN-38 is an active metabolic form of CPT-11 that is one of the most promising anti-cancer drugs. Membrane vesicles prepared from cells expressing dmL0MRP1 could transport SN-38, but not LTC4 or estradiol-17 (beta-D-glucuronate), and could not be photolabeled with azidoAG-A. These data suggested that SN-38 was transported by a different mechanism than that of GSH-dependent transport. Understanding the GSH-independent transport mechanism of MRP1, and identification of drugs that are transported by this mechanism, will be critical for combating MRP1-mediated drug resistance. We performed a pairwise comparison of compounds that are transported by MRP1 in a GSH-dependent or -independent manner. These data indicated that it may be possible to predict compounds that are transported by MRP1 in a GSH-independent manner.     

Formation and disposition of diethylphosphoryl-obidoxime, a potent anticholinesterase that is hydrolyzed by human paraoxonase (PON1)

The potential of pyridinium-4-aldoximes, such as obidoxime, to reactivate diethylphosphorylated acetylcholinesterases is not fully exploited due to the inevitable formation of phosphoryloximes (POX) with high anticholinesterase activity. Mono(diethylphosphoryl) obidoxime (DEP-obidoxime) was isolated for the first time showing remarkable stability under physiological conditions (half-life 13.5min; pH 7.1; 37 degrees C). The half-life was considerably extended to 20h at 0 degrees C, which facilitated the preparation and allowed isolation by HPLC. The structure was confirmed by mass spectrometry and the degradation pattern. DEP-obidoxime decomposed by an elimination reaction forming the intermediate nitrile that hydrolyzed mainly into the pyridone and cyanide. The intermediates were prepared and confirmed by mass spectroscopy. DEP-Obidoxime was an extremely potent inhibitor of human acetylcholinesterase approaching a second-order rate constant of 10(9)M(-1)min(-1) (pH 7.4; 37 degrees C). The nitrile and the pyridone were still good reactivators. In the presence of human plasma DEP-obidoxime was hydrolyzed into parent obidoxime. Calcium-dependence and sensitivity towards chelators, substitution pattern by other divalent cations and protein-modifying agents all pointed to human paraoxonase (hPON1) as the responsible protein with POX-hydrolase activity. Subjects, probably belonging to the homozygous (192)arginine subtype, were virtually devoid of POX-hydrolase activity while a highly purified hPON1 of the homozygous (192)glutamine subtype exhibited particularly high POX-hydrolase activity. Two parathion-poisoned patients with high and low POX-hydrolase activity responded well and poorly, respectively, to obidoxime treatment although the former patient had higher plasma paraoxon levels than the poor responder. Hence, the POX-hydrolase associated PON1 subtype may be another contributor that modulates pyridinium-4-aldoxime effectiveness.     

The polymorphic human glutathione transferase T1-1, the most efficient glutathione transferase in the denitrosation and inactivation of the anticancer drug 1,3-bis(2-chloroethyl)-1-nitrosourea

A member of the Theta class of human glutathione transferases (GST T1-1) was found to display the greatest catalytic activity towards the cytostatic drug 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) of the GSTs studied. In this investigation (the most extensive to date), enzymes from four classes of the soluble human GSTs were heterologously expressed, purified, and kinetically characterized. From the 12 enzymes examined, only GST M2-2, GST M3-3 and GST T1-1 had significant activities with BCNU. This establishes that the activity is not a characteristic of a particular class of GSTs. Although GST M3-3 was previously reported to have the greatest activity with BCNU, the current investigation demonstrates that GST M2-2 is equally active and that GST T1-1 has an approximately 20-fold higher specific activity than either of the Mu class enzymes. A more rigorous kinetic analysis of GST T1-1 gave the following parameters with BCNU: a k(cat) of 0.035 +/-0.003s(-1) and a K(M) of 1.0 +/- 0.1mM. The finding that GST T1-1 has the highest activity towards BCNU is significant since GST T1-1 is expressed in the brain, a common target for BCNU treatment. Furthermore, the existence of a GST T1-1 null allele in up to 60% in some populations, may influence both the sensitivity of tumors to chemotherapy and the severity of adverse side-effects in patients treated with this agent.     

Evaluation of the binding of the tricyclic isoxazole photoaffinity label LY475776 to multidrug resistance associated protein 1 (MRP1) orthologs and several ATP- binding cassette (ABC) drug transporters

Several of the ATP-binding cassette (ABC) transporters confer resistance to anticancer agents and/or antiviral agents when overexpressed in drug-sensitive cells. Recently a MRP1 (ABCC1) tricyclic isoxazole inhibitor, LY475776 was shown to be a glutathione-dependent photoaffinity label of human MRP1 and showed poor labeling of murine mrp1, an ortholog that does not confer anthracycline resistance. In the present study, the specificity of LY475776 was examined for its ability to modulate or photolabel orthologs of MRP1 and several other drug efflux transporters of the ABC transporter family. LY475776 modulated MRP1 and Pgp-mediated resistance (MDR, ABCB1) in, respectively, HeLa-T5 and CEM/VLB(100) cells to both vincristine and doxorubicin. LY475776 photolabeled 170kDa Pgp and was inhibited by the potent Pgp inhibitor LY335979 (Zosuquidar.3HCl). The labeling of the 190kDa MRP1 protein in membranes of HeLa-T5 cells was inhibited by substrates of MRP1 such as leukotriene C(4), vincrisine, and doxorubicin and by the inhibitor, MK571. LY475776 did not photolabel human MRP2 (ABCC2), MRP3 (ABCC3), MRP5 (ABCC5) or breast cancer resistance protein (ABCG2). Because LY475776 photolabels murine mrp1 less well than human MRP1 and binds to a region believed important for anthracycline binding, studies were conducted with monkey and canine MRP1 which also show a reduced ability to confer resistance to anthracyclines. Unlike murine mrp1, both orthologs were photolabeled well by LY475776. These studies indicate that the specificity of LY475776 is fairly limited to Pgp and MRP1 and further studies will help to define the binding regions.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) induces oxidative stress, DNA strand breaks, and poly(ADP-ribose) polymerase-1 activation in human breast carcinoma cell lines

The formation of reactive oxygen species (ROS) plays a critical role in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced toxicities in mammalian cells since it promotes cell proliferation, growth arrest, and apoptosis. In this study, we investigated whether TCDD induces oxidative stress and DNA damage in human ERalpha(+)/MCF-7 and ERalpha(-)/MDA-MB-231 breast cancer cells and whether this is accompanied by the initiation of DNA repair events. Results indicated that viability of MCF-7 and MDA-MB-231 cells was concentration- and time-dependently reduced by TCDD. Further, we observed significant increases in ROS formation and decreases in intracellular glutathione (GSH) in these two cell lines after TCDD treatment. Overall, the extent of cell death was greater in MCF-7 cells than in MDA-MB-231 cells whereas the magnitude of ROS formation and GSH depletion was greater in MDA-MB-231 cells than in MCF-7 cells. In addition, we observed that at non-cytotoxic concentration (1nM for 5h), TCDD induced decreases in intracellular NAD(P)H and NAD(+) in MCF-7 and MDA-MB-231 cells. These decreases were completely blocked by three types of poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors. The catalytic activation of PARP-1 in cells treated with TCDD was confirmed by detection of the presence of polymers of ADP-ribose-modified PARP-1 using Western blotting. Moreover, we demonstrated increases in the number of DNA strand breaks in MCF-7 and MDA-MB-231 cells exposed to TCDD as measured by the single-cell gel electrophoresis (Comet) assay. Overall, this evidence confirms that TCDD induces decreases in intracellular NAD(P)H and NAD(+) through PARP-1 activation mediated by formation of DNA strand breaks. In addition, we demonstrated that the extent of oxidative stress and DNA damage was greater in MDA-MB-231 cells than in MCF-7 cells, with a strong correlation to estrogen receptor (ER) status. In conclusions, our findings add further support to the theme that ROS formation is a significant determinant factor in mediating the induction of oxidative DNA damage and repair in human breast cancer cells exposed to TCDD and that the TCDD-induced oxidative stress and DNA damage may, in part, contribute to TCDD-induced carcinogenesis.     

Role of two adjacent cytoplasmic tyrosine residues in MRP1 (ABCC1) transport activity and sensitivity to sulfonylureas

The human ATP-binding cassette (ABC) protein MRP1 causes resistance to many anticancer drugs and is also a primary active transporter of conjugated metabolites and endogenous organic anions, including leukotriene C(4) (LTC(4)) and glutathione (GSH). The sulfonylurea receptors SUR1 and SUR2 are related ABC proteins with the same domain structure as MRP1, but serve as regulators of the K(+) channel Kir6.2. Despite their functional differences, the activity of both SUR1/2 and MRP1 can be blocked by glibenclamide, a sulfonylurea used to treat diabetes. Residues in the cytoplasmic loop connecting transmembrane helices 15 and 16 of the SUR proteins have been implicated as molecular determinants of their sensitivity to glibenclamide and other sulfonylureas. We have now investigated the effect of mutating Tyr(1189) and Tyr(1190) in the comparable region of MRP1 on its transport activity and sulfonylurea sensitivity. Ala and Ser substitutions of Tyr(1189) and Tyr(1190) caused a > or =50% decrease in the ability of MRP1 to transport different organic anions, and a decrease in LTC(4) photolabeling. Kinetic analyses showed the decrease in GSH transport was attributable primarily to a 10-fold increase in K(m). In contrast, mutations of these Tyr residues had no major effect on the catalytic activity of MRP1. Furthermore, the mutant proteins showed no substantial differences in their sensitivity to glibenclamide and tolbutamide. We conclude that MRP1 Tyr(1189) and Tyr(1190), unlike the corresponding residues in SUR1, are not involved in its differential sensitivity to sulfonylureas, but nevertheless, may be involved in the transport activity of MRP1, especially with respect to GSH.     

Isolation and identification of heterogeneous nuclear ribonucleoproteins (hnRNP) from purified plasma membranes of human tumour cell lines as albumin-binding proteins

Since albumin is being developed as a drug carrier to target tumours the search for albumin-binding proteins (ABPs), which play a role in cell surface binding and endocytosis of native and conjugated albumins becomes more and more interesting. We isolated five different proteins from purified plasma membranes from three different human tumour cell lines (CCRF-CEM, MV3 and MCF7) by albumin affinity chromatography and identified them as four members of the heterogeneous nuclear ribonucleoproteins (hnRNP) family and calreticulin by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry. Contamination of the plasma membrane preparation by nuclear membranes was excluded with anti-nucleopore antibodies. Western blot analyses of plasma membranes showed ABPs with the same molecular weights as the albumin-affinity isolates. Tryptic digestion of intact cells was used to determine the sidedness of the albumin-binding property, which is oriented to the exterior of the cell. Localisation to the plasma membrane and albumin binding is a novel property of hnRNP.     

A new flavone glycoside: 5,7,3',4'-tetrahydroxy-3-methoxy flavone-7-O-beta-D-galactopyranosyl-(1-->4)-O-beta-D-glucopyranoside from the stem of Acacia catechu Willd

A new bio-active flavone glycoside, m.p. C 28 H 32 O 17 , mp 283-284°C, M + 640 [EIMS] was isolated from the ethylacetate soluble fraction of the ethanolic extract of the stems of Acacia catechu and its structure was characterised as 5,7,3',4'-tetrahydroxy-3-methoxy flavone-7- O - β- d -galactopyranosyl-(1 →4)- O - β- d -glucopyranoside by various chemical degradations and spectral analyses.     

Wogonin and fisetin induce apoptosis in human promyeloleukemic cells, accompanied by a decrease of reactive oxygen species, and activation of caspase 3 and Ca(2+)-dependent endonuclease

Seven structurally related flavonoids including luteolin, nobiletin, wogonin, baicalein, apigenin, myricetin and fisetin were used to study their biological activities on the human leukemia cell line, HL-60. On MTT assay, wogonin, baicalein, apigenin, myricetin and fisetin showed obvious cytotoxic effects on HL-60 cells, with wogonin and fisetin being the most-potent apoptotic inducers among them. The cytotoxic effects of wogonin and fisetin were accompanied by the dose- and time-dependent appearance of characteristics of apoptosis including DNA fragmentation, apoptotic bodies and the sub-G1 ratio. Treatment with an apoptosis-inducing concentration of wogonin or fisetin causes rapid and transient induction of caspase 3/CPP32 activity, but not caspase 1 activity. Further, cleavage of poly(ADP-ribose) polymerase (PARP) and decrease of pro-caspase 3 protein were detected in wogonin- and fisetin-treated HL-60 cells. An increase in the pro-apoptotic protein, bax, and a decrease in the anti-apoptotic protein, Mcl-1, were detected in fisetin- and wogonin-treated HL-60 cells. However, Bcl-2, Bcl-XL, and Bad all remained unchanged in wogonin- and fisetin-treated HL-60 cells. In vitro chromatin digestion revealed that endonuclease activity was profoundly enhanced in wogonin- and fisetin-treated HL-60 cells, and the addition of ethylenediaminetetraacetic acid (EDTA) or ethyleneglycoltetraacetic acid (EGTA) into the reaction blocked endonuclease activation and at an optimum pH of 7.5. The caspase 3 inhibitor, Ac-DEVD-CHO, but not the caspase 1 inhibitor, Ac-YVAD-CHO, attenuated wogonin- and fisetin-induced DNA ladders, PARP cleavage, and endonuclease activation. Pretreatment of HL-60 cells with N-acetyl-cysteine or catalase efficiently inhibited H(2)O(2) (200 microM)-induced apoptosis, but showed no inhibitory effect on wogonin- and fisetin-induced DNA ladders, caspase 3 activation, or bax protein induction. Decrease in endogenous ROS production was detected in wogonin- and fisetin-treated HL-60 cells by DCHF-DA assay. In conclusion, our experiments indicate that a decrease in intracellular peroxide level was involved in wogonin- and fisetin-induced apoptosis; activation of caspase 3 and endonuclease, induction of bax protein and suppression of Mcl-1 protein were detected in the process.     

Preparation and characterization of dialkylphosphoryl-obidoxime conjugates, potent anticholinesterase derivatives that are quickly hydrolyzed by human paraoxonase (PON1192Q)

The potential of the most active pyridinium-4-aldoximes, such as obidoxime and trimedoxime, to reactivate phosphorylated acetylcholinesterase is not fully exploited because of inevitable formation of phosphoryloximes (POXs) with extremely high anticholinesterase activity. Hence, a topochemical equilibrium is expected at the active site, with the freshly reactivated enzyme being rapidly re-inhibited by POX produced during reactivation. In the present study, dimethylphosphoryl-, diethylphosphoryl-, and diisopropyl-obidoxime conjugates were generated and isolated in substance. Their inhibition rate of acetylcholinesterase from human red cell membranes was by a factor of 2250, 480 and 600 higher than that observed with paraoxon-methyl, paraoxon-ethyl, and diisopropyl phosphorofluoridate, respectively. All three POXs were hydrolyzed by human paraoxonase (PON1), with the alloenzyme PON1192Q being about 50-fold more active than PON1192R. The rate of hydrolysis, yielding obidoxime, was 1:6:0.03 for the three POXs, respectively. The rate of non-enzymic degradation, yielding obidoxime mononitrile, was similar with the three POXs and showed a high dependency on the reaction temperature (activation energy 83 kJ/mol), while enzymic hydrolysis required less energy (16 kJ/mol). To determine POX-hydrolase activity, we preferred a reaction temperature of 20 degrees C to reduce the noise of spontaneous degradation. A plot of POX-hydrolase versus salt-stimulated paraoxonase activity showed a highly discriminating power towards the PON1Q192R alloenzymes, which may be based on repulsive forces of the quaternary nitrogen atoms of the protonated arginine subtype and the bisquaternary POXs. It is concluded that the pharmacogenetic PON1Q192R polymorphism may be another contributor to the large variability of susceptible subjects seen in obidoxime-treated patients.     

Perchlorate transport and inhibition of the sodium iodide symporter measured with the yellow fluorescent protein variant YFP-H148Q/I152L

Perchlorate is an environmental contaminant that impairs thyroid function by interacting with the sodium iodide symporter (NIS), the transporter responsible for iodide uptake in the thyroid gland. Perchlorate is well known as a competitive inhibitor of iodide transport by NIS, and recent evidence demonstrates that NIS can also transport perchlorate. In this study, we evaluated the yellow fluorescent protein (YFP) variant YFP-H148Q/I152L, as a genetically encodable biosensor of intracellular perchlorate concentration monitored by real-time fluorescence microscopy. Fluorescence of recombinant YFP-H148Q/I152L was suppressed by perchlorate and iodide with similar affinities of 1.2 mM and 1.6 mM, respectively. Perchlorate suppressed YFP-H148Q/I152L fluorescence in FRTL-5 thyroid cells and NIS-expressing COS-7 cells, but had no effect on COS-7 cells lacking NIS. Fluorescence changes in FRTL-5 cells were Na⁺-dependent, consistent with the Na⁺-dependence of NIS activity. Perchlorate uptake in FRTL-5 cells resulted in 10-fold lower intracellular concentrations than iodide uptake, and was characterized by a higher affinity (K_m 4.6 μM for perchlorate and 34.8 μM for iodide) and lower maximal velocity (V_max 6.8 μM/s for perchlorate and 39.5 μM/s for iodide). Perchlorate also prevented iodide-induced changes in YFP-H148Q/I152L fluorescence in FRTL-5 cells, with half-maximal inhibition occurring at 1.1-1.6 μM. In conclusion, YFP-H148Q/I152L detects perchlorate accumulation by thyroid and other NIS-expressing cells, and reveals differences in the kinetics of perchlorate versus iodide transport by NIS.     

Mass spectrometric analysis of 4-hydroxy-1-(3-pyridyl)-1-butanone-releasing DNA adducts in human lung

An improved analytical method was developed for the analysis of 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB)-releasing DNA adducts in lung samples of patients undergoing surgery for lung cancer. HPB-releasing adducts can be formed by metabolic activation of the tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and N'-nitrosonornicotine, and have been reported to play an important role in tobacco carcinogenesis. [2,2,3,3-D(4)]HPB (D(4)-HPB) was used as an internal standard, and HPB released by acid hydrolysis of DNA was determined by gas chromatography/mass spectrometry in the negative ion chemical ionisation mode. The method is sensitive with a limit of detection of 5.9 fmol HPB and a limit of quantification of 15.2 fmol HBP/mg DNA. The recovery of HPB was 82+/-17% and the background response was 10.1+/-1.8 fmol HPB/sample. The concentration of HPB-releasing lung DNA adducts was significantly higher (p<0.0001) in 21 self-reported smokers compared to in 11 self-reported nonsmokers (404+/-258 fmol versus 59+/-56 fmol HPB/mg DNA, respectively). HPB-releasing hemoglobin adduct concentrations were only marginally higher in a subset of 12 smokers compared to in 7 nonsmokers (63+/-53 fmol versus 42+/-34 fmol HPB/g hemoglobin; p=0.36). No correlation was found between HPB-releasing adducts in DNA and hemoglobin (p=0.074).     

The accumulation and efflux of lead partly depend on ATP-dependent efflux pump–multidrug resistance protein 1 and glutathione in testis Sertoli cells

Since lead accumulation is toxic to cells, its excretion is crucial for organisms to survive the toxicity. In this study, mouse testis sertoli (TM4) and Mrp1 lower-expression TM4-sh cells were used to explore the lead accumulation characteristics, and the role of ATP-dependent efflux pump–multidrug resistance protein 1 (Mrp1) in lead excretion. TM4 cells possess Mrp-like transport activity. The expression levels of mrp1 mRNA and Mrp1 increased after lead treatments at first and then decreased. The maximum difference of relative mRNA expression reached 10 times. In the presence of lead acetate, the amount of cumulative lead in TM4-sh was much higher than that in TM4. After the treatment with lead acetate at 10–40 μM for 12 h or 24 h, the differences were about 2–8 times. After with the switch to lead-free medium, the cellular lead content in TM4-sh remains higher than that in TM4 cells at 1,3, 6, and 9 h time points (P < 0.01). Energy inhibitor sodium azide, Mrp inhibitors MK571 and glutathione (GSH) biosynthesis inhibitor BSO could block lead efflux from TM4 cells significantly. These results indicate that lead excretion may be mediated by Mrp1 and GSH in TM4 cells. Mrp1 could be one of the important intervention points for lead detoxification.     

Reductive activation of the prodrug 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(4-nitrophenyl)ethoxy]carbonyl]hydrazine (KS119) selectively occurs in oxygen-deficient cells and overcomes O-alkylguanine-DNA alkyltransferase mediated KS119 tumor cell resistance

1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(4-nitrophenyl)ethoxy]carbonyl]hydrazine (KS119) is a prodrug of the 1,2-bis(sulfonyl)hydrazine class of antineoplastic agents designed to exploit the oxygen-deficient regions of cancerous tissue. Thus, under reductive conditions in hypoxic cells this agent decomposes to produce the reactive intermediate 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine (90CE), which in turn generates products that alkylate the O⁶-position of guanine in DNA. Comparison of the cytotoxicity of KS119 in cultured cells lacking O⁶-alkylguanine-DNA alkyltransferase (AGT) to an agent such as Onrigin™, which through base catalyzed activation produces the same critical DNA G-C cross-link lesions by the generation of 90CE, indicates that KS119 is substantially more potent than Onrigin™ under conditions of oxygen deficiency, despite being incompletely activated. In cell lines expressing relatively large amounts of AGT, the design of the prodrug KS119, which requires intracellular activation by reductase enzymes to produce a cytotoxic effect, results in an ability to overcome resistance derived from the expression of AGT. This appears to derive from the ability of a small portion of the chloroethylating species produced by the activation of KS119 to slip through the cellular protection afforded by AGT to generate the few DNA G-C cross-links that are required for tumor cell lethality. The findings also demonstrate that activation of KS119 under oxygen-deficient conditions is ubiquitous, occurring in all of the cell lines tested thus far, suggesting that the enzymes required for reductive activation of this agent are widely distributed in many different tumor types.     

Nicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone induce cyclooxygenase-2 activity in human gastric cancer cells: Involvement of nicotinic acetylcholine receptor (nAChR) and beta-adrenergic receptor signaling pathways

Induction of cyclooxygenase-2 (COX-2) associates with cigarette smoke exposure in many malignancies. Nicotine and its derivative, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), are the two important components in cigarette smoke that contributes to cancer development. However, the molecular mechanism(s) by which nicotine or NNK promotes gastric carcinogenesis remains largely unknown. We found that nicotine and NNK significantly enhanced cell proliferation in AGS cells that expressed both alpha7 nicotinic acetylcholine receptor (α7 nAChR) and β-adrenergic receptors. Treatment of cells with α-bungarotoxin (α-BTX, α7nAChR antagonist) or propranolol (β-adrenergic receptor antagonist) blocked NNK-induced COX-2/PGE₂ and cell proliferation, while nicotine-mediated cell growth and COX-2/PGE₂ induction can only be suppressed by propranolol, but not α-BTX. Moreover, in contrast to the dependence of growth promoting effect of nicotine on Erk activation, inhibitor of p38 mitogen-activated protein kinase (MAPK) repressed NNK-induced COX-2 upregulation and resulted in suppression of cell growth. In addition, nicotine and NNK mediated COX-2 induction via different receptors to modulate several G1/S transition regulatory proteins and promote gastric cancer cell growth. Selective COX-2 inhibitor (SC-236) caused G1 arrest and abrogated nicotine/NNK-induced cell proliferation. Aberrant expression of cyclin D1 and other G1 regulatory proteins are reversed by blockade of COX-2. These results pointed to the importance of adrenergic and nicotinic receptors in gastric tumor growth through MAPK/COX-2 activation, which may perhaps provide a chemoprevention strategy for cigarette smoke-related gastric carcinogenesis.