Functional assay and structure-activity relationships of new third-generation P-glycoprotein inhibitors

Twenty-eight compounds, including 24 structurally related derivatives of tariquidar synthesized in our laboratory, and four XR compounds, reported by Xenova group Ltd, were investigated by the Hoechst 33342 and Calcein AM functional assays for estimation of their inhibitory effects on the transport activity of P-glycoprotein (P-gp). A high correlation between the effects obtained in both assays was observed at the substrate concentrations used. The analyses of kinetics data from experiments at different substrate concentrations revealed non-competitive inhibition in the Calcein AM assay and competitive inhibition in the Hoechst 33342 assay. The 3D structures of the compounds were further aligned on Hoechst 33342 using flexible and pharmacophore alignments. The results suggested that inhibitors could interact with the H-binding site of P-gp and this could potentially be achieved by different ways of binding. The best 3D-QSAR models, generated by CoMFA and CoMSIA, yielded an internal predictive squared correlation coefficient higher than 0.8 and included electrostatic, steric, hydrogen bond acceptor, and hydrophobic fields. Validation of the models on an external test set of 30 XR compounds gave predictive squared correlation coefficients of up to 0.66. An excellent correspondence between the experimental and modeled activities of the test compounds was observed. The models can be used for prediction and rational design of new P-gp inhibitors.     

Investigation of chalcones and benzochalcones as inhibitors of breast cancer resistance protein

Breast cancer resistance protein (BCRP/ABCG2) belongs to the ATP binding cassette family of transport proteins. BCRP has been found to confer multidrug resistance in cancer cells. A strategy to overcome resistance due to BCRP overexpression is the investigation of potent and specific BCRP inhibitors. The aim of the current study was to investigate different multi-substituted chalcones for their BCRP inhibition. We synthesized chalcones and benzochalcones with different substituents (viz. OH, OCH(3), Cl) on ring A and B of the chalcone structure. All synthesized compounds were tested by Hoechst 33342 accumulation assay to determine inhibitory activity in MCF-7 MX and MDCK cells expressing BCRP. The compounds were also screened for their P-glycoprotein (P-gp) and Multidrug resistance-associated protein 1 (MRP1) inhibitory activity in the calcein AM accumulation assay and were found to be selective towards inhibition of BCRP. Substituents at position 2' and 4' on chalcone ring A were found to be essential for activity; additionally there was a great influence of substituents on ring B. Presence of 3,4-dimethoxy substitution on ring B was found to be optimal, while presence of 2- and 4-chloro substitution also showed a positive effect on BCRP inhibition.     

Screening Compounds with a Novel High-Throughput ABCB1-Mediated Efflux Assay Identifies Drugs with Known Therapeutic Targets at Risk for Multidrug Resistance Interference

ABCB1, also known as P-glycoprotein (P-gp) or multidrug resistance protein 1 (MDR1), is a membrane-associated multidrug transporter of the ATP-binding cassette (ABC) transporter family. It is one of the most widely studied transporters that enable cancer cells to develop drug resistance. Reliable high-throughput assays that can identify compounds that interact with ABCB1 are crucial for developing new therapeutic drugs. A high-throughput assay for measuring ABCB1-mediated calcein AM efflux was developed using a fluorescent and phase-contrast live cell imaging system. This assay demonstrated the time- and dose-dependent accumulation of fluorescent calcein in ABCB1-overexpressing KB-V1 cells. Validation of the assay was performed with known ABCB1 inhibitors, XR9576, verapamil, and cyclosporin A, all of which displayed dose-dependent inhibition of ABCB1-mediated calcein AM efflux in this assay. Phase-contrast and fluorescent images taken by the imaging system provided additional opportunities for evaluating compounds that are cytotoxic or produce false positive signals. Compounds with known therapeutic targets and a kinase inhibitor library were screened. The assay identified multiple agents as inhibitors of ABCB1-mediated efflux and is highly reproducible. Among compounds identified as ABCB1 inhibitors, BEZ235, BI 2536, IKK 16, and ispinesib were further evaluated. The four compounds inhibited calcein AM efflux in a dose-dependent manner and were also active in the flow cytometry-based calcein AM efflux assay. BEZ235, BI 2536, and IKK 16 also successfully inhibited the labeling of ABCB1 with radiolabeled photoaffinity substrate [¹²⁵I]iodoarylazidoprazosin. Inhibition of ABCB1 with XR9576 and cyclosporin A enhanced the cytotoxicity of BI 2536 to ABCB1-overexpressing cancer cells, HCT-15-Pgp, and decreased the IC₅₀ value of BI 2536 by several orders of magnitude. This efficient, reliable, and simple high-throughput assay has identified ABCB1 substrates/inhibitors that may influence drug potency or drug-drug interactions and predict multidrug resistance in clinical treatment.     

Structure-activity relationships of new inhibitors of breast cancer resistance protein (ABCG2)

At the end of the last century tariquidar (XR9576) was synthesized, pharmacologically investigated, and classified as a promising 3rd generation P-glycoprotein (P-gp) modulator. Following the discovery of BCRP in 1998 an increasing number of substances were studied in relation to their potency to interact with this transporter. Recently it has been shown that XR9576 inhibits both P-gp and BCRP transport function similarly to GF120918 (elacridar). This observation prompted us to investigate 5 XR compounds and 25 structurally related derivatives synthesized in our laboratory for their BCRP inhibitory effect. The biological activity data were determined by our new Hoechst 33342 assay that has been transferred from P-gp to BCRP overexpressing cells. 3D-QSAR models (CoMFA and CoMSIA) were generated and validated by the leave-many-out method and the scrambling stability test. The best models yielded an internal predictive squared correlation coefficient higher than 0.8 and contained steric, electrostatic, hydrophobic, and hydrogen bond donor fields. To our knowledge, this is the first 3D-QSAR analysis of BCRP inhibitors. Additionally the biological activity data determined in P-gp overexpressing cells on one side and BCRP overexpressing cells on the other side were compared to identify selective and non-selective inhibitors of P-gp and BCRP. The results may help to get a better insight which structural elements are necessary to direct the interaction of these compounds with P-gp and/or BCRP.     

Synthesis and biological evaluation of a small molecule library of 3rd generation multidrug resistance modulators

The development of new modulators possessing high efficacy, low toxicity and high selectivity is a pivotal approach to overcoming P-glycoprotein (P-gp) mediated multidrug resistance (MDR) in tumour cells. In this study 39 compounds are presented which have been synthesized and pharmacologically investigated in our laboratory. Similarly to the potent 3rd generation MDR modulator tariquidar (XR9576) the compounds contain a tetrahydroisoquinoline–ethyl-phenylamine substructure that, in contrast to XR9576, is connected to a smaller hydrophobic part, thus leading to molecules of lower molecular weight. The connection between the tetrahydroisoquinoline–ethyl-phenylamine substructure and the hydrophobic part was achieved through four different types of linkers: amide, urea, amide-ether and amide-styryl. A number of structural modifications in the hydrophobic part were created. The calcein AM assay served as test system to determine the P-gp transport inhibitory potencies of the compounds. For the amide linker derivatives a structure–activity relationship analysis was performed outlining which structural modifications contributed to the inhibitory potency. The compounds containing a bicyclic hydrophobic part with a particular substituent in a specific orientation were identified as the most potent amide derivatives. Among the urea derivatives the compounds with highest inhibitory potency possessed an ortho-nitro substituent. The conformational analysis revealed that this position enables the formation of a hydrogen bond to the urea linker thus stabilizing the conformation. Regarding the amide-styryl derivatives the elongation of the amide linker seemed to be most decisive for the observed increase in activity. The most promising candidate in the whole library possess an amide-ether linker and an ortho-nitro substituent in the hydrophobic part. This compound inhibites P-gp slightly less than tariquidar and can serve as a lead structure for new potent P-gp modulators.     

Proton motive force-dependent Hoechst 33342 transport by the ABC transporter LmrA of Lactococcus lactis

The fluorescent compound Hoechst 33342 is a substrate for many multidrug resistance (MDR) transporters and is widely used to characterize their transport activity. We have constructed mutants of the adenosine triphosphate (ATP) binding cassette (ABC)-type MDR transporter LmrA of Lactococcus lactis that are defective in ATP hydrolysis. These mutants and wild-type LmrA exhibited an atypical behavior in the Hoechst 33342 transport assay. In membrane vesicles, Hoechst 33342 transport was shown to be independent of the ATPase activity of LmrA, and it was not inhibited by orthovanadate but sensitive to uncouplers that collapse the proton gradient and to N,N'-dicyclohexylcarbodiimide, an inhibitor of the F0F1-ATPase. In contrast, transport of Hoechst 33342 by the homologous, heterodimeric MDR transporter LmrCD showed a normal ATP dependence and was insensitive to uncouplers of the proton gradient. With intact cells, expression of LmrA resulted in an increased rate of Hoechst 33342 influx while LmrCD caused a decrease in the rate of Hoechst 33342 influx. Cellular toxicity assays using a triple knockout strain, i.e., L. lactis delta lmrA delta lmrCD, demonstrate that expression of LmrCD protects cells against the growth inhibitory effects of Hoechst 33342, while in the presence of LmrA, cells are more susceptible to Hoechst 33342. Our data demonstrate that the LmrA-mediated Hoechst 33342 transport in membrane vesicles is influenced by the transmembrane pH gradient due to a pH-dependent partitioning of Hoechst 33342 into the membrane.     

Methanethiosulfonate derivatives of rhodamine and verapamil activate human P-glycoprotein at different sites

The human multidrug resistance P-glycoprotein (P-gp, ABCB1) actively extrudes a broad range of potentially cytotoxic compounds out of the cell. Key steps in understanding the transport process are binding of drug substrates in the transmembrane domains, initiation of ATPase activity, and subsequent drug efflux. We used cysteine-scanning mutagenesis of the transmembrane segment residues and reaction with the thiol-reactive drug substrate analog of rhodamine, methane-thiosulfonate-rhodamine (MTS-rhodamine), to test whether P-gp could be trapped in an activated state with high levels of ATPase activity. The presence of such an activated P-gp could be used to further investigate P-gp-drug substrate interactions. Single cysteine mutants (149) were treated with MTS-rhodamine, and ATPase activities were determined after removal of unreacted MTS-rhodamine. One mutant, F343C(TM6), showed a 5.8-fold increase in activity after reaction with MTS-rhodamine. Pre-treatment of mutant F343C with rhodamine B protected it from activation by MTS-rhodamine, indicating that residue Cys-343 contributes to the rhodamine-binding site. The ATPase activity of MTS-rhodamine-treated mutant F343C, however, was not stimulated further by colchicine or calcein-AM. By contrast, verapamil and Hoechst 33342 stimulated and inhibited, respectively, the ATPase activity of the MTS-rhodamine-treated mutant F343C. These results indicate that the MTS-rhodamine binding site overlaps that of colchicine and calcein-AM but not that of verapamil and Hoechst 33342 within the common drug-binding pocket.     

Modulation of function of three ABC drug transporters, P-glycoprotein (ABCB1), mitoxantrone resistance protein (ABCG2) and multidrug resistance protein 1 (ABCC1) by tetrahydrocurcumin, a major metabolite of curcumin

Many studies have been performed with the aim of developing effective resistance modulators to overcome the multidrug resistance (MDR) of human cancers. Potent MDR modulators are being investigated in clinical trials. Many current studies are focused on dietary herbs due to the fact that these have been used for centuries without producing any harmful side effects. In this study, the effect of tetrahydrocurcumin (THC) on three ABC drug transporter proteins, P-glycoprotein (P-gp or ABCB1), mitoxantrone resistance protein (MXR or ABCG2) and multidrug resistance protein 1 (MRP1 or ABCC1) was investigated, to assess whether an ultimate metabolite form of curcuminoids (THC) is able to modulate MDR in cancer cells. Two different types of cell lines were used for P-gp study, human cervical carcinoma KB-3-1 (wild type) and KB-V-1 and human breast cancer MCF-7 (wild type) and MCF-7 MDR, whereas, pcDNA3.1 and pcDNA3.1-MRP1 transfected HEK 293 and MXR overexpressing MCF7AdrVp3000 or MCF7FL1000 and its parental MCF-7 were used for MRP1 and MXR study, respectively. We report here for the first time that THC is able to inhibit the function of P-gp, MXR and MRP1. The results of flow cytometry assay indicated that THC is able to inhibit the function of P-gp and thereby significantly increase the accumulation of rhodamine and calcein AM in KB-V-1 cells. The result was confirmed by the effect of THC on [³H]-vinblastine accumulation and efflux in MCF-7 and MCF-7MDR. THC significantly increased the accumulation and inhibited the efflux of [³H]-vinblastine in MCF-7 MDR in a concentration-dependent manner. This effect was not found in wild type MCF-7 cell line. The interaction of THC with the P-gp molecule was clearly indicated by ATPase assay and photoaffinity labeling of P-gp with transport substrate. THC stimulated P-gp ATPase activity and inhibited the incorporation of [¹²⁵I]-iodoarylazidoprazosin (IAAP) into P-gp in a concentration-dependent manner. The binding of [¹²⁵I]-IAAP to MXR was also inhibited by THC suggesting that THC interacted with drug binding site of the transporter. THC dose dependently inhibited the efflux of mitoxantrone and pheophorbide A from MXR expressing cells (MCF7AdrVp3000 and MCF7FL1000). Similarly with MRP1, the efflux of a fluorescent substrate calcein AM was inhibited effectively by THC thereby the accumulation of calcein was increased in MRP1-HEK 293 and not its parental pcDNA3.1-HEK 293 cells. The MDR reversing properties of THC on P-gp, MRP1, and MXR were determined by MTT assay. THC significantly increased the sensitivity of vinblastine, mitoxantrone and etoposide in drug resistance KB-V-1, MCF7AdrVp3000 and MRP1-HEK 293 cells, respectively. This effect was not found in respective drug sensitive parental cell lines. Taken together, this study clearly showed that THC inhibits the efflux function of P-gp, MXR and MRP1 and it is able to extend the MDR reversing activity of curcuminoids in vivo.     

Comparison of 3 assay systems using a common probe substrate, calcein AM, for studying P-gp using a selected set of compounds

The multidrug resistance protein 1 (MDR1) transporter is the most abundantly investigated adenosine triphosphate (ATP)-Binding Cassette (ABC) transporter protein. Multiple assay systems were developed to study MDR1-mediated transport and possible drug-drug interactions. Yet, as different probe substrates are used in these assays, it is difficult to directly compare the results. In this study, a common probe substrate was applied in 3 assay systems developed to study MDR1: the cellular dye efflux assay, the ATPase assay, and the vesicular transport assay. This probe substrate is calcein acetoxymethyl ester (calcein AM), the acetoxymethyl ester derivative of the fluorescent dye, calcein. Using a common probe allows the investigation of the effect of passive permeability on the result obtained by testing various compounds. In this study, 22 compounds with different logP values were tested in the above-mentioned 3 assay systems. The vesicular transport assay proved most sensitive, detecting 18 of 22 interactions with the protein. The ATPase assay detected 15 interactions, whereas the cellular dye efflux assay was the least sensitive with only 10 hits. A correlation was found between the hydrophobicity of the compound and the ratio of cellular and vesicular transport IC(50) values, indicating the effect of passive permeability on the result. Based on hydrophobicity, the current study provides guidelines on applying the most correct tool for studying MDR1 interactions.     

Microfluorometric Evaluation of Calcein Acetoxymethyl Ester as a Probe for P-Glycoprotein-Mediated Resistance: Effects of Cyclosporin A and Its Nonimmunosuppressive Analogue SDZ PSC 833

A microtiter plate-based fluorometric assay for functional measurement of 170-kDa P-glycoprotein (Pgp)-mediated transport using fluorescent calcein as a probe is described. The myeloma RPMI 8226 cell line and two of its doxorubicin-resistant Pgp-expressing sublines, dox40 (high expression) and dox6 (low expression), were used as models. Nonfluorescent calcein acetoxymethyl ester (calcein/AM) was added to the cells and subsequent accumulation of calcein was measured in a 96-well scanning fluorometer after 30 min. There was an inverse relationship between Pgp expression and calcein/AM accumulation, which increased dose-dependently in the presence of cyclosporin A (CsA) and the nonimmunosuppressive analogue SDZ PSC 833 (PSC) in the Pgp-expressing cell lines. PSC appeared to restore uptake more effectively than CsA at low concentrations. Calcein accumulation was also increased in Pgp-expressing cells by the addition of the Pgp substrate vincristine and the metabolic inhibitor potassium cyanide, KCN. No effect was observed in parental cell lines. When parental and dox40 cells were mixed, 10% of dox40 cells could reproducibly be detected. The results indicate that microtiter-plate determination of calcein accumulation is a simple and sensitive method for functional determination of Pgp-mediated drug transport. The method may become useful, not only for preclinical screening for novel and improved resistance modifiers, but also for determination of Pgp activity in individual clinical tumor samples.     

Estradiol attenuation of β-amyloid-induced toxicity: A comparison of MTT and calcein AM assays

17β-estradiol (βE2) has been shown to attenuate the toxicity of β-amyloid peptides (Aβ) in neuronal cultures with the effective concentration of βE2 ranging from low nM to high μM. This study compares the effective neuroprotective concentration of βE2 against both Aβ-mediated toxicity in a human neuroblastoma cell line, SK-N-SH using cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as an endpoint to the effective βE2 concentration obtained using a calcein acetoxymethyl ester (calcein AM) viability assay. The minimum βE2 concentration required for protection varied 1000-fold between the two viability assays with 1 nM βE2 conferring significant protection in the calcein AM assay but 1 μM βE2 required for significant protection in the MTT assay. Interestingly, the maximal inhibition of MTT reduction occured at sub-toxic Aβ concentrations and did not correlate with other markers of cellular viability including calcein fluorescence, dye exclusion (propidium iodide or trypan blue), cellular ATP levels, or reduction of another tetrazolium dye, 5-(3-carboxymethoxyphenyl)-2-(4,5-dimethylthiazolyl)-3-(4-sulfophenyl) tetrazolium (MTS). By contrast, there was no difference between the MTT and calcein AM assays with respect to H₂O₂ toxicity or the neuroprotective effectiveness of 10 nM βE2 against H_{2 2} toxicity. These results indicate that low concentrations of βE2 can attenuate Aβ and H₂O₂ toxicity in a human neuroblastoma cell line. Further, these results suggest that the MTT assay is not an appropriate assay for the determination of βE2-mediated attenuation of Aβ toxicity.     

Drug binding in human P-glycoprotein causes conformational changes in both nucleotide-binding domains

The human multidrug resistance P-glycoprotein (P-gp, ABCB1) uses ATP to transport many structurally diverse compounds out of the cell. It is an ABC transporter with two nucleotide-binding domains (NBDs) and two transmembrane domains (TMDs). Recently, we showed that the "LSGGQ" motif in one NBD ((531)LSGGQ(535) in NBD1; (1176)LSGGQ(1180) in NBD2) is adjacent to the "Walker A" sequence ((1070)GSSGCGKS(1077) in NBD2; (427)GNSGCGKS(434) in NBD1) in the other NBD (Loo, T. W., Bartlett, M. C., and Clarke, D. M. (2002) J. Biol. Chem. 277, 41303-41306). Drug substrates can stimulate or inhibit the ATPase activity of P-gp. Here, we report the effect of drug binding on cross-linking between the LSGGQ signature and Walker A sites (Cys(431)(NBD1)/C1176C(NBD2) and Cys(1074)(NBD2)/L531C(NBD1), respectively). Seven drug substrates (calcein-AM, demecolcine, cis(Z)-flupentixol, verapamil, cyclosporin A, Hoechst 33342, and trans(E)-flupentixol) were tested for their effect on oxidative cross-linking. Substrates that stimulated the ATPase activity of P-gp (calcein-AM, demecolcine, cis(Z)-flupentixol, and verapamil) increased the rate of cross-linking between Cys(431)(NBD1-Walker A)/C1176C(NBD2-LSGGQ) and between Cys(1074)(NBD2-Walker A)/L531C(NBD1-LSGGQ) when compared with cross-linking in the absence of drug substrate. By contrast, substrates that inhibited ATPase activity (cyclosporin A, Hoechst 33342, and trans(E)-flupentixol) decreased the rate of cross-linking. These results indicate that interaction between the LSGGQ motifs and Walker A sites must be essential for coupling drug binding to ATP hydrolysis. Drug binding in the transmembrane domains can induce long range conformational changes in the NBDs, such that compounds that stimulate or inhibit ATPase activity must decrease and increase, respectively, the distance between the Walker A and LSGGQ sequences.     

Inhibitory effect of phospholipids on P-glycoprotein: Cellular studies in Caco-2, MDCKII mdr1 and MDCKII wildtype cells and P-gp ATPase activity measurements

Phospholipids are widely used excipients for pharmaceutical formulations, such as for preparing biphasic systems or to solubilize or encapsulate poorly soluble drugs. The present study investigates a new property of this class of substance: its ability to inhibit the efflux transporter Pglycoprotein (P-gp). P-gp is expressed in the intestinal epithelium, thereby significantly impairing the systemic absorption of various pharmaceutically active substances. The phospholipid screening performed in this study involved derivatives with different headgroups and fatty acid residues and a number of experimental parameters. For in vitro studies we carried out transport experiments and calcein accumulation assays in Caco-2- and MDCKII mdr1 and wildtype cell lines. The three compounds which displayed significant P-gp inhibition in both assays and in Caco-2 as well as in MDCKII mdr1, consisted of phosphatidylcholine (PC) and either two saturated fatty acid residues of eight (8:0 PC) or ten carbon atoms (10:0 PC), or of two unsaturated docosahexaeonic acid residues (cis-22:6 PC).Supported by P-gp ATPase activity measurements, 8:0 and 10:0 PC were assumed to function as direct P-gp inhibitors interacting with the transporter probably in their monomeric state, whereas a different, as yet unknown mechanism of action applied for cis-22:6 PC.Because of their proven ability to significantly inhibit P-gp in vitro, these phospholipids shall further be elucidated in vivo, whether they may truly serve to increase the bioavailability of orally applied drugs with a P-gp substrate character.     

Calcein AM release-based cytotoxic cell assay for fish leucocytes

A non-specific cytotoxic cell assay for fish is presented that is based on the release of the activated fluorochrome calcein AM from lysed carp epithelioma papulosum cyprini (EPC) cells. To establish the suitability of treating EPC cells with calcein AM the uptake and spontaneous release of the calcein AM by the EPC cells was evaluated. Incubation of 5 microM calcein AM in culture medium with 1x10(5)EPC cells well(-1)for a minimum of 3 h provided sufficient labelling. Spontaneous release of fluorescence from the labelled EPC cells during 10 h of post labelling incubation ranged from 30 to 39% of the total observed fluorescence. Cytotoxic activity of trout leucocytes was evaluated at three leucocyte to target cell ratios (10:1, 2:1 and 1:1) following incubation (4, 6, 8, and 10 h) with calcein AM-labelled EPC cells at 15 degrees C. In some instances, the monoclonal antibody specific for the NCC surface receptor NCCRP-1 (MAb5C.6) was included in the cultures. The activity of NCC cells was significantly inhibited in the presence of 0.25 microg well(-1)of MAb5C.6 relative to no antibody (P相似文献   

In vitro activity of novel dual action MDR anthranilamide modulators with inhibitory activity on CYP-450 (Part 2)

Synthesis and in vitro cytotoxicity assays of new anthranilamide MDR modulators have been performed to assess their inhibition potency on the P-glycoprotein (P-gp) transporter. Previous studies showed that the replacement of the aromatic spacer group between nitrogen atoms (N(1) and N(2)) in the P-gp inhibitor XR9576 with ethyl or propyl chain is optimal for P-gp inhibition potency. To confirm that observation, the ethyl or the propyl linker arm was replaced with a pyrrolidine or an alicyclic group such as cyclohexyl. In addition, an arylpiperazinyl group and two methoxyl groups onto the anthranilic part were introduced to assess their effect on the anti P-gp activity. Five molecules were prepared and evaluated on CEM/VLB500. All new anthranilamides were more potent than verapamil, most of them exhibited a lower cytotoxicity than XR9576. Compound 5 was the most potent and its inhibition activity was similar to XR9576. Interestingly, in vitro biotransformation studies of compounds 4 and 5 using human CYP-450 isoforms revealed, that conversely to XR9576, compounds 4 and 5 inhibited CYP3A4, an enzyme that colocalizes with P-gp in the intestine and contributes to tumor cell chemoresistance by enhancing the biodisposition of numerous drugs, notably paclitaxel. In that context, 5 might be suitable for further drug development.     

Effect of drug efflux blockers on vital staining of cellular DNA with Hoechst 33342

The present study shows that staining of certain live cells, e.g., adriamycin-resistant P388 cells, by Hoechst 33342 is difficult because of the presence of a rapid efflux pump, which reduces intracellular dye concentration. Coincubation of these refractory cells in the presence of efflux blockers such as phenothiazines (trifluoperazine) or Ca++ channel blockers (verapamil) enhances dye retention and thus leads to generation of normal DNA distribution histograms. Laser flow cytometric data is confirmed by fluorometric assays, which show that P388/R cells retain one-third the amount of Hoechst 33342, and coincubation with efflux blockers increases Hoechst retention to values similar to those of drug-sensitive P388 cells. DNA histograms of mouse splenocytes incubated with Hoechst 33342 alone have a bimodal distribution possibly because of the presence of subpopulations that do not retain the fluorochrome owing to rapid efflux. Coincubation with an efflux blocker results in the generation of unimodal DNA histograms from these cells. These preliminary studies suggest that reduced retention of Hoechst 33342 in certain cell types (because of rapid efflux) can be blocked by efflux blockers, thus leading to generation of typical DNA distribution histograms.     

Design,synthesis and biological evaluation of lapachol derivatives possessing indole scaffolds as topoisomerase I inhibitors

A series of novel lapachol derivatives possessing indole scaffolds was designed and synthesized. The in vitro anti-proliferative activity of these novel compounds was evaluated in Eca109 and Hela cell lines. Almost all the tested compounds showed manifested potent inhibitory activity against the two tested cancer cell lines. Topo I-mediated DNA relaxation activity indicated that these novel compounds have potent Topoisomerase I inhibition activity. The most potent compounds 4n and 4k demonstrated more cytotoxicity than camptothecin and was comparable to camptothecin in inhibitory activities on Topoisomerase I in our biological assay. In addition, the Hoechst 33342 staining method also showed that the complex can induce Hela cell apoptosis.     

Combined localization and real-time functional studies using a GFP-tagged ABCG2 multidrug transporter

ABCG2 is a half-transporter which causes multidrug resistance when overexpressed in tumor cells. Availability of combined localization and functional assays would greatly improve cell biology and drug modulation studies for this transporter. Here we demonstrate that an N-terminally GFP-tagged version of the protein (GFP-G2) can be used to directly monitor ABCG2 expression, dimerization, localization and function in living cells. GFP-G2 is fully functional when tested for drug-stimulated ATPase activity, vesicular transport assay, subcellular localization or cell surface epitope conformational changes. By measuring both GFP and Hoechst 33342 dye fluorescence in HEK-293 cells, we provide evidence that a real-time transport assay can be reliably applied to identify ABCG2 substrates, transport modulators, as well as to monitor the cellular functions of this multidrug transporter protein. This approach also avoids the need of cloning, drug selection or other further separation or characterization of the transgene-expressing cells.     

Kinetic analyses as a critical parameter in defining the side population (SP) phenotype

The side population (SP) phenotype has been reported as a method to identify hematopoietic stem cells in the bone marrow based upon differential staining with the fluorescent dye, Hoechst 33342. This technique has drawn great interest in the stem cell community, as it may provide a simple approach to the enrichment of progenitor cells from a variety of normal and malignant tissues. The frequency of these cells and their performance in functional assays has varied considerably within the literature. To investigate mechanisms that may contribute to the SP phenotype, we measured the fluorescence emission of Hoechst-stained bone marrow cells as a function of both time and dye concentration using a custom flow cytometer and data acquisition software. These measurements demonstrate that all nucleated cells within the bone marrow undergo an identical staining pattern at varying rates, even under conditions previously reported to abrogate the SP. Therefore, the SP phenotype is not unique to stem cells, but rather represents a transient feature of marrow cells exposed to Hoechst 33342 for varying amounts of time. We propose that heterogeneity of SP-defined populations may be a consequence of the rate at which differing cell populations accumulate Hoechst 33342. Further, we suggest that dye uptake kinetics will likely be an important factor for optimal use of Hoechst 33342 in isolating stem cells.     

哺乳动物精子质量的评价方法

精子的各种功能状态反应了精子的受精能力。检测精子质膜完整性的荧光探针有SYBR－14,SYTO－17,CFDA、CDMFDA、CAM、PI、Hoechst33258、Hoechst33342,其中以SYBR－14结合PI使用效果最好,检测线粒体活性的荧光探针有JC－1、MITO、Rh123,JC-1比MITO和Rh123更适用于检测精子线粒体功能,检测顶体状态的荧光探针有PNA－FITC、PSA－FITC、LYSO－G及CTC等。检测获能状态的荧光探针有CTC。此外,还可以通过检测精子与透明带的结合能力、精子穿入去透明带卵子的能力以及使卵子受精的能力和其后胚胎的发育能力等方面来评价精子的功能状态。