Phosphinanes and Azaphosphinanes as Potent and Selective Inhibitors of Activated Thrombin-Activatable Fibrinolysis Inhibitor (TAFIa).

Selective and potent inhibitors of activated thrombin activatable fibrinolysis inhibitor (TAFIa) have the potential to increase endogenous and therapeutic fibrinolysis and to behave like profibrinolytic agents without the risk of major hemorrhage, since they do not interfere either with platelet activation or with coagulation during blood hemostasis. Therefore, TAFIa inhibitors could be used in at-risk patients for the treatment, prevention, and secondary prevention of stroke, venous thrombosis, and pulmonary embolisms. In this paper, we describe the design, the structure-activity relationship (SAR), and the synthesis of novel, potent, and selective phosphinanes and azaphosphinanes as TAFIa inhibitors. Several highly active azaphosphinanes display attractive properties suitable for further in vivo efficacy studies in thrombosis models.

A far-red fluorescent probe based on a phospha-fluorescein scaffold for cytosolic calcium imaging.

The far-red emissive fluorescent probe CaPF-1 based on a phospha-fluorescein scaffold enables the detection of cytosolic calcium ions in living cells. The probe can be excited in the red region (λabs = 636 nm) and exhibits a sufficiently high fluorescence turn-on response in the far-red region (λem = 663 nm) upon complexation with calcium ions. The hydrophilic and anionic characteristics of this phospha-fluorescein fluorophore allowed the cytosolic localization of CaPF-1. Moreover, it was possible to visualize histamine-induced calcium oscillation in HeLa cells using CaPF-1.

Phostine PST3.1a Targets MGAT5 and Inhibits Glioblastoma-Initiating Cell Invasiveness and Proliferation.

Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor and accounts for a significant proportion of all primary brain tumors. Median survival after treatment is around 15 months. Remodeling of N-glycans by the N-acetylglucosamine glycosyltransferase (MGAT5) regulates tumoral development. Here, perturbation of MGAT5 enzymatic activity by the small-molecule inhibitor 3-hydroxy-4,5-bis-benzyloxy-6-benzyloxymethyl-2-phenyl2-oxo-2λ5-[1,2]oxaphosphinane (PST3.1a) restrains GBM growth. In cell-based assays, it is demonstrated that PST3.1a alters the ß1,6-GlcNAc N-glycans of GBM-initiating cells (GIC) by inhibiting MGAT5 enzymatic activity, resulting in the inhibition of TGFßR and FAK signaling associated with doublecortin (DCX) upregulation and increase oligodendrocyte lineage transcription factor 2 (OLIG2) expression. PST3.1a thus affects microtubule and microfilament integrity of GBM stem cells, leading to the inhibition of GIC proliferation, migration, invasiveness, and clonogenic capacities. Orthotopic graft models of GIC revealed that PST3.1a treatment leads to a drastic reduction of invasive and proliferative capacity and to an increase in overall survival relative to standard temozolomide therapy. Finally, bioinformatics analyses exposed that PST3.1a cytotoxic activity is positively correlated with the expression of genes of the epithelial-mesenchymal transition (EMT), while the expression of mitochondrial genes correlated negatively with cell sensitivity to the compound. These data demonstrate the relevance of targeting MGAT5, with a novel anti-invasive chemotherapy, to limit glioblastoma stem cell invasion. Mol Cancer Res; 15(10); 1376-87. ©2017 AACR.

Inhibition of glycolysis by a novel EGFR/HER2 inhibitor KU004 suppresses the growth of HER2+ cancer.

Upregulation of glycolysis was often observed in human HER2-overexpressing cancers. In this study, we demonstrated that KU004, a dual novel EGFR/HER2 inhibitor, disrupted cancer cell proliferation via modulation of glycolysis. KU004, inhibited the Warburg effect by suppressing hexokinase II (HK2) expression at the transcriptional and post-translational levels. Further study demonstrated that the downregulation of HKII by KU004 was mainly mediated by the PI3K/Akt signaling pathway. Furthermore, the role of HKII downregulation in KU004-mediated antitumor effect was also confirmed in our in vivo xenograft model. Collectively, these data suggest that multifaceted targeting the aberrant glucose metabolism along with the upstream HER2 may be an effective approach for clinical treatment against HER2+ cancer.

Molecular and structural basis for the roles of hepatitis C virus polymerase NS5B amino acids 15, 223, and 321 in viral replication and drug resistance.

Resistance to the 2'-F-2'-C-methylguanosine monophosphate nucleotide hepatitis C virus (HCV) inhibitors PSI-352938 and PSI-353661 was associated with a combination of amino acid changes (changes of S to G at position 15 [S15G], C223H, and V321I) within the genotype 2a nonstructural protein 5B (NS5B), an RNA-dependent RNA polymerase. To understand the role of these residues in viral replication, we examined the effects of single and multiple point mutations on replication fitness and inhibition by a series of nucleotide analog inhibitors. An acidic residue at position 15 reduced replicon fitness, consistent with its proximity to the RNA template. A change of the residue at position 223 to an acidic or large residue reduced replicon fitness, consistent with its proposed proximity to the incoming nucleoside triphosphate (NTP). A change of the residue at position 321 to a charged residue was not tolerated, consistent with its position within a hydrophobic cavity. This triple resistance mutation was specific to both genotype 2a virus and 2'-F-2'-C-methylguanosine inhibitors. A crystal structure of the NS5B S15G/C223H/V321I mutant of the JFH-1 isolate exhibited rearrangement to a conformation potentially consistent with short primer-template RNA binding, which could suggest a mechanism of resistance accomplished through a change in the NS5B conformation, which was better tolerated by genotype 2a virus than by viruses of other genotypes.

KU004 induces G1 cell cycle arrest in human breast cancer SKBR-3 cells by modulating PI3K/Akt pathway.

KU004 is a newly synthesized compound which has been demonstrated possessing potent anti-cancer activities through targeting the highly-expressed protein HER2 on the surface of the cells. In this study, we investigated the potential roles of KU004 in the induced-cell cycle arrest in human breast cancer SK-BR-3 cells. KU004 could not only inhibit the proliferation of SK-BR-3 in a concentration-dependent manner but also induce G1 phase arrest in SK-BR-3 cells. The western blot results showed KU004 decreased the expression of cyclin D, CDK-4, p-Rb708/780, and up-regulated the p21. In order to verify whether KU004 takes the anti-tumor effect thought the regulation of PI3K/Akt pathway, we used western blot to detect the expression of protein Akt, Her2, p-Akt and p-Her2. Our results shown that after KU004 treatment, the amount of p-Akt and p-Her2 decreased but the total amount of Akt and Her2 remained unchanged. In conclusion, these results provide a framework for further exploration of KU004 as a novel chemotherapeutic for human breast tumors by modulating PI3K/Akt pathway.

Oxaphosphinanes: new therapeutic perspectives for glioblastoma.

This paper reports the design and the synthesis of a new family of compounds, the phostines, belonging to the [1,2]oxaphosphinane family. Twenty-six compounds have been screened for their antiproliferative activity against a large panel of NCI cancer cell lines. Because of its easy synthesis and low EC(50) value (500 nM against the C6 rat glioma cell line), compound 3.1a was selected for further biological study. Moreover, the specific biological effect of 3.1a on the glioblastoma phylogenetic cluster from the NCI is dependent on its stereochemistry. Within that cluster, 3.1a has a higher antiproliferative activity than Temozolomide and is more potent than paclitaxel for the SF295 and SNB75 cell lines. In constrast with paclitaxel and vincristine, 3.1a is devoid of astrocyte toxicity. The original activity spectrum of 3.1a on the NCI cancer cell line panel allows the development of this family for use in association with existing drugs, opening new therapeutic perspectives.

Synthesis of stable isotope labeled analogs of the anti-hepatitis C virus nucleotide prodrugs PSI-7977 and PSI-352938.

In order to support bioanalytical LC/MS method development and plasma sample analysis in preclinical and clinical studies of the anti-hepatitis C-virus nucleotides, PSI-7977 and PSI-352938, the corresponding stable isotope labeled forms were prepared. These labeled compounds were prepared by addition reaction of the freshly prepared Grignard reagent (13)CD(3)MgI to the corresponding 2 '-ketone nucleosides followed by fluorination of the resulting carbinol with DAST. As expected, these 2 '-C-(trideuterated-(13)C-methyl) nucleotide prodrugs showed similar anti-HCV activity to that of the corresponding unlabeled ones.

Hepatitis C virus nucleotide inhibitors PSI-352938 and PSI-353661 exhibit a novel mechanism of resistance requiring multiple mutations within replicon RNA.

PSI-352938, a cyclic phosphate nucleotide, and PSI-353661, a phosphoramidate nucleotide, are prodrugs of ß-D-2'-deoxy-2'-α-fluoro-2'-ß-C-methylguanosine-5'-monophosphate. Both compounds are metabolized to the same active 5'-triphosphate, PSI-352666, which serves as an alternative substrate inhibitor of the NS5B RNA-dependent RNA polymerase during HCV replication. PSI-352938 and PSI-353661 retained full activity against replicons containing the S282T substitution, which confers resistance to certain 2'-substituted nucleoside/nucleotide analogs. PSI-352666 was also similarly active against both wild-type and S282T NS5B polymerases. In order to identify mutations that confer resistance to these compounds, in vitro selection studies were performed using HCV replicon cells. While no resistant genotype 1a or 1b replicons could be selected, cells containing genotype 2a JFH-1 replicons cultured in the presence of PSI-352938 or PSI-353661 developed resistance to both compounds. Sequencing of the NS5B region identified a number of amino acid changes, including S15G, R222Q, C223Y/H, L320I, and V321I. Phenotypic evaluation of these mutations indicated that single amino acid changes were not sufficient to significantly reduce the activity of PSI-352938 and PSI-353661. Instead, a combination of three amino acid changes, S15G/C223H/V321I, was required to confer a high level of resistance. No cross-resistance exists between the 2'-F-2'-C-methylguanosine prodrugs and other classes of HCV inhibitors, including 2'-modified nucleoside/-tide analogs such as PSI-6130, PSI-7977, INX-08189, and IDX-184. Finally, we determined that in genotype 1b replicons, the C223Y/H mutation failed to support replication, and although the A15G/C223H/V321I triple mutation did confer resistance to PSI-352938 and PSI-353661, this mutant replicated at only about 10% efficiency compared to the wild type.

Antinociceptive effect of cyclic phosphatidic acid and its derivative on animal models of acute and chronic pain.

BACKGROUND: Cyclic phosphatidic acid (cPA) is a structural analog of lysophosphatidic acid (LPA), but possesses different biological functions, such as the inhibition of autotaxin (ATX), an LPA-synthesizing enzyme. As LPA is a signaling molecule involved in nociception in the peripheral and central systems, cPA is expected to possess analgesic activity. We characterized the effects of cPA and 2-carba-cPA (2ccPA), a chemically stable cPA analog, on acute and chronic pain. RESULTS: (1) The systemic injection of 2ccPA significantly inhibited somato-cardiac and somato-somatic C-reflexes but not the corresponding A-reflexes in anesthetized rats. (2) 2ccPA reduced sensitivity measured as the paw withdrawal response to electrical stimulation applied to the hind paws of mice through the C-fiber, but not Aδ or Aß. (3) In mice, pretreatment with 2ccPA dose-dependently inhibited the second phase of formalin-induced licking and biting responses. (4) In mice, pretreatment and repeated post-treatments with 2ccPA significantly attenuated thermal hyperalgesia and mechanical allodynia following partial ligation of the sciatic nerve. (5) In rats, repeated post-treatments with 2ccPA also significantly attenuated thermal hyperalgesia and mechanical allodynia following chronic sciatic nerve constriction. CONCLUSIONS: Our results suggest that cPA and its stable analog 2ccPA inhibit chronic and acute inflammation-induced C-fiber stimulation, and that the central effects of 2ccPA following repeated treatments attenuate neuropathic pain.

Stereoselective synthesis of PSI-352938: a ß-D-2'-deoxy-2'-α-fluoro-2'-ß-C-methyl-3',5'-cyclic phosphate nucleotide prodrug for the treatment of HCV.

PSI-352938 is a novel 2'-deoxy-2'-α-fluoro-2'-ß-C-methyl 3',5'-cyclic phosphate nucleotide prodrug currently under investigation for the treatment of hepatitis C virus (HCV) infection. PSI-352938 demonstrated superior characteristics in vitro that include broad genotype coverage, superior resistance profile, and high levels of active triphosphate in vivo in the liver compared to our first and second generation nucleoside inhibitors of this class. Consequently, PSI-352938 was selected for further development and an efficient and scalable synthesis was sought to support clinical development. We report an improved, diastereoselective synthesis of a key 1'-ß-nucleoside intermediate 13 via S(N)2 displacement of 1-α-bromo ribofuranose sugar 16 with the potassium salt of 6-chloro-2-amino purine and an efficient method to prepare cis-Rp cyclic phosphate (PSI-352938) in a highly stereoselective manner without any chromatographic purification. The 1-α-bromo sugar 16 was stereospecifically prepared from the corresponding 1-ß-lactol in high yield under mild bromination conditions using CBr(4)/PPh(3) (Appel reaction). The desired cis-Rp 3',5'-cyclic phosphate construction was accomplished using isopropyl phosphorodichloridate readily obtained from POCl(3) and isopropyl alcohol. The base combination of Et(3)N/NMI was identified as a key factor for producing PSI-352938 as the major (>95%) diastereomer (cis-Rp) in high yield after the final cyclization step. The current route described in this article was successfully used to produce PSI-352938 on multikilogram scale.

Inhibition of hepatitis C virus replicon RNA synthesis by PSI-352938, a cyclic phosphate prodrug of ß-D-2'-deoxy-2'-α-fluoro-2'-ß-C-methylguanosine.

PSI-352938 is a novel cyclic phosphate prodrug of ß-D-2'-deoxy-2'-α-fluoro-2'-ß-C-methylguanosine 5'-monophosphate that has potent activity against hepatitis C virus (HCV) in vitro. The studies described here characterize the in vitro anti-HCV activity of PSI-352938, alone and in combination with other inhibitors of HCV, and the cross-resistance profile of PSI-352938. The effective concentration required to achieve 50% inhibition for PSI-352938, determined using genotype 1a-, 1b-, and 2a-derived replicons stably expressed in the Lunet cell line, were 0.20, 0.13, and 0.14 µM, respectively. The active 5'-triphosphate metabolite, PSI-352666, inhibited recombinant NS5B polymerase from genotypes 1 to 4 with comparable 50% inhibitory concentrations. In contrast, PSI-352938 did not inhibit the replication of hepatitis B virus or human immunodeficiency virus in vitro. PSI-352666 did not significantly affect the activity of human DNA and RNA polymerases. PSI-352938 and its cyclic phosphate metabolites did not affect the cyclic GMP-mediated activation of protein kinase G. Clearance studies using replicon cells demonstrated that PSI-352938 cleared cells of HCV replicon RNA and prevented replicon rebound. An additive to synergistic effect was observed when PSI-352938 was combined with other classes of HCV inhibitors, including alpha interferon, ribavirin, NS3/4A inhibitors, an NS5A inhibitor, and nucleoside/nucleotide and nonnucleoside inhibitors. Cross-resistance studies showed that PSI-352938 remained fully active against replicons containing the S282T or the S96T/N142T amino acid alteration. Replicons that contain mutations conferring resistance to various classes of nonnucleoside inhibitors also remained sensitive to inhibition by PSI-352938. PSI-352938 is currently being evaluated in a phase I clinical study in genotype 1-infected individuals.

Development and pharmacologic characterization of deoxybromophospha sugar derivatives with antileukemic activity.

Here, we synthesized two phospha sugar derivatives, 2,3,4-tribromo-3-methyl-1-phenylphospholane 1-oxide (TMPP) and 2,3-dibromo-3-methyl-1-phenylphospholane 1-oxide (DMPP) by reacting 3-methyl-1-phenyl-2-phospholene 1-oxide with bromine, and investigated their potential as antileukemic agents in cell lines. Both agents showed inhibitory effects on leukemia cell proliferation, with mean IC(50) values of 6.25 micromol/L for TMPP and 23.7 micromol/L for DMPP, indicating that inhibition appeared to be dependent on the number of bromine atoms in the structure. Further, TMPP at 10 micromol/L and DMPP at 20 micromol/L induced G2/M cell cycle block in leukemia cells, and TMPP at 20 micromol/L induced apoptosis in these cells. TMPP treatment effected a reduction in both cell cycle progression signals (FoxM1, KIS, Cdc25B, Cyclin D1, Cyclin A, and Aurora-B) and tumor cell survival (p27(Kip1) and p21(Cip1)), as well as induced the activation of caspase-3 and -9. Further, treatment with TMPP significantly reduced the viability of AML specimens derived from AML patients, but only slightly reduced the viability of normal ALDH(hi) progenitor cells. We also observed that FoxM1 mRNA was overexpressed in AML cells, and treatment with TMPP reduced FoxM1 mRNA expression in AML cells. Here, we report on the synthesis of TMPP and DMPP and demonstrate that these agents hinder proliferation of leukemia cells by FoxM1 suppression, which leads to G2/M cell cycle block and subsequent caspase-3-dependent apoptosis in acute leukemia cells. These agents may facilitate the development of new strategies in targeted antileukemic therapy.

Ability of carbazole salts, inhibitors of Alzheimer beta-amyloid fibril formation, to cross cellular membranes.

Alzheimer's disease is characterized by the presence of beta-amyloid fibril formation. The inhibition of this peptide accumulation may be a prevention method for Alzheimer's disease. Several classes of molecules have been reported to inhibit beta-amyloid fibril formation and among them carbazoles. However, very few studies have been performed to determine the destination of such molecules in vivo and especially if they can pass the blood brain barrier. The aim of this paper is to study whether carbazoles could pass the blood brain barrier, i.e. if they can circumvent ATP Binding Cassette (ABC) transporters such as P-glycoprotein (P-gp) and Multidrug Resistance-associated protein (MRP1) which efficiently limit drug brain uptake. For this purpose we have synthesized a fluorescent derivative of carbazole benzothiazolium iodide 1,2 disubstituted ethylene (referred as carbazole thiazole: CT), which can be easily detected and followed in the pre-trial study phases in cells or in tissue. We use cellular models overexpressing P-gp and MRP1. Our results show that: i) CT is able to cross membranes and to penetrate rapidly inside the cells, ii) CT is a P-gp substrate and consequently its accumulation in P-gp overexpressing cells is very low, iii) CT is a poor MRP1 substrate. In addition once inside the cells, CT rapidly binds to DNA and is then slowly reduced by intracellular reducing agents. In conclusion, the efficiency of carbazole derivatives in inhibiting the beta-amyloid formation in vivo could be highly compromised because, as P-gp substrates, they will probably not cross the blood brain barrier.

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.

5-Carboxamido-1,3,2-dioxaphosphorinanes, potent inhibitors of MTP.

5-Carboxamido-1,3,2-dioxaphosphorinanes have been identified as potent inhibitors of microsomal triglyceride-transfer protein. The 1,3,2-dioxaphosphorine functionality acted as a neutral and stable replacement for piperidine and piperidine N-oxide.

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.

MRP1 modulation by PAK-104P: detection with technetium-99m-MIBI in cultured lung tumor cells.

BACKGROUND: Resistance mediated by the MultiDrug Resistance protein (P-glycoprotein and MRP1), results in energy-dependent efflux of drugs and 99mTc-MIBI from the cells. The goal of our investigation was to evaluate the capacity of PAK-104P to lower multidrug resistance by decreasing substrate efflux. MATERIALS AND METHODS: 99mTc-MIBI accumulation was quantified in the leukaemia cell line which expresses the P-glycoprotein (K562/R) or not (K562/S) and the small lung cancer cell line which expresses MRP1 (GLC4/R) or not (GLC4/S). Three experimental protocols were used: 1). cells were treated with increasing concentrations of PAK-104P; 2). the plasma membrane potential was lowered; 3). intracellular reduced glutathione (GSH) was depleted. RESULTS: Exposure of cells to 5 microM PAK-104P affected 99mTc-MIBI accumulation as follow: 1). no effect on K562 cell lines; 2). increased 8-fold in GLC4/R; 3). enhanced in GLC4 after GSH concentration and transmembrane potential reduction. CONCLUSION: Assessed by 99mTc-MIBI, PAK-104P modulated MRP1 activity by the decrease of intracellular GSH concentration.

Kinetics of glutathione and daunorubicin efflux from multidrug resistance protein overexpressing small-cell lung cancer cells.

The present study examined how the multidrug resistance protein (MRP1), which is an ATP-dependent anionic conjugate transporter, also mediates the transport of reduced glutathione (GSH) and the co-transport of the cationic drug, daunorubicin, with GSH in living GLC4/Adr cells. To obtain information on the affinity of GSH for the multidrug resistance protein in GLC4/Adr cells, we investigated the GSH concentration dependence of the ATP-dependent GSH efflux. The intracellular GSH concentration was modulated by preincubation of the cells with 25 microM buthionine sulfoximine, an inhibitor of GSH synthetase, for 0-24 h. The transport of GSH was related to the intracellular GSH concentration up to approximately 5 mM and then plateaued. Fitting of the obtained data according to the Michaelis-Menten equation revealed a Km of 3.4+/-1.4 mM and a Vmax of 1.5+/-0.2x10(-18) mol/cell/s. The ATP-dependent transport of GSH was inhibited by 3-([[3-(2-[7-chloro-2-quinolinyl]ethenyl)phenyl]-[(3-dimethylamino-3-oxopropyl)-thio]-methyl]thio)propanoic acid (MK571), with 50% inhibition being obtained with 1.4 microM MK571. We investigated the GSH concentration dependence of the MRP1-mediated ATP-dependent transport of daunorubicin under conditions where the transport of daunorubicin became saturated. The daunorubicin transport was related to the intracellular GSH concentration up to approximately 5 mM and then plateaued. We were therefore in the situation where GSH acted as an activator: its presence was necessary for the binding and transport of daunorubicin by MRP1. However, GSH was also transported by the multidrug resistance protein. The concentration of GSH that gave half the maximal rate of daunorubicin efflux was 2.1+/-0.8 mM, very similar to the Km value obtained for GSH. In conclusion, the rate of daunorubicin efflux, under conditions where the transport of daunorubicin became saturated, and the rate of GSH efflux determined at any intracellular concentration of GSH were very similar, yielding a 1:1 stoichiometry with respect to GSH and daunorubicin transport. These results support a model in which daunorubicin is co-transported with GSH.

Reversal of LRP-associated drug resistance in colon carcinoma SW-620 cells.

Resistance to multiple drugs is mediated by lung resistance-related protein (LRP) as well as P-glycoprotein (P-gp) and multidrug resistance protein (MRP). The levels of expression of LRP mRNA and LRP in a human colon carcinoma cell line, SW-620, were increased by the differentiation-inducing agent, sodium butyrate (NaB). Treatment of SW-620 cells with NaB for 2 weeks conferred resistance to adriamycin (ADM) and VP-16. The resistance was almost completely reversed by PAK-104P, a pyridine analog, but not by cepharanthine. ADM accumulated mainly in the nuclei of SW-620 cells not treated with NaB and in the cytoplasm of SW-620 cells treated with NaB. When the NaB-treated SW-620 cells were incubated with ADM in the presence of PAK-104P, the accumulation of ADM in nuclei was substantially increased. Isolated nuclei from untreated cells accumulated more ADM than nuclei from NaB-treated cells. Efflux of ADM from the nuclei isolated from NaB-treated cells was enhanced. PAK-104P and an antibody against LRP increased the accumulation of ADM in the isolated nuclei from NaB-treated cells, and inhibited the enhanced efflux of ADM from the nuclei. These findings suggest that at least in part, PAK-104P reverses LRP-mediated drug resistance by inhibiting the efflux of ADM from nuclei. PAK-104P may be useful for reversing MDR in tumors that overexpress LRP.