ABCB1 Efflux Research Articles

Tivozanib reverses multidrug resistance mediated by ABCB1 (P-glycoprotein) and ABCG2 (BCRP)

This study aimed to investigate the mechanism of reversal of multidrug resistance mediated by ABC transporters with tivozanib (AV-951 and KRN-951). Tivozanib is a potent inhibitor of VEGF-1, -2 and -3 receptors. ABCB1- and ABCG2-overexpressing cell lines were treated with respective substrate antineoplastic agents in the presence or absence of tivozanib. The results indicate that tivozanib can significantly reverse ABCB1-mediated resistance to paclitaxel, vinblastine and colchicine, as well as ABCG2-mediated resistance to mitoxantrone, SN-38 and doxorubicin. Drug efflux assays showed that tivozanib increased the intracellular accumulation of substrates by inhibiting the ABCB1 and ABCG2 efflux activity. Furthermore, at a higher concentration, tivozanib inhibited the ATPase activity of both ABCB1 and ABCG2 and inhibited the photolabeling of ABCB1 or ABCG2. We conclude that tivozanib at noncytotoxic concentrations has the previously unknown activity of reversing multidrug resistance mediated by ABCB1 and ABCG2 transporters.

Development and validation of an ultra-high performance LC-MS/MS assay for intracellular SN-38 in human solid tumour cell lines: comparison with a validated HPLC-fluorescence method.

A simple and rapid ultra-high performance liquid chromatography-mass spectrometry/mass spectrometry (UPLC-MS/MS) method has been developed for measuring intracellular concentrations of the anticancer agent 7-ethyl-10-hydroxycamptothecin (SN-38) in tumour cells using camptothecin (CPT) as internal standard. SN-38 extraction was carried out using acidified acetonitrile. SN-38 and CPT were separated on a PFP column using gradient elution with acidified water and acetonitrile. SN-38 and CPT were quantified using a triple quadrupole mass spectrometry system. Least square regression calibration lines were obtained with average correlation coefficients of R(2)=0.9993±0.0016. The lower limit of detection (LOD) and lower limit of quantification (LOQ) for SN-38 were 0.1 and 0.3ng/ml, respectively. CPT recovery was 98.5±13% and SN-38 recoveries at low quality control (LQC, 5ng/ml) and high quality control (HQC, 500ng/ml) were 89±6% and 95±8%, respectively. The intra- and inter-day imprecision for LQC was 5.8 and 8.5%, and for HQC was 6.3 and 4.4%, respectively. The method was compared to a validated high performance liquid chromatography-fluorescent method. In addition, the method has been successfully applied to determine the intracellular accumulation of SN-38 investigating the transport through ABCB1 (P-gp) and ABCG2 (BCRP) efflux pumps in colorectal cancer cell lines.

Identification and characterization of the molecular interactions of persistent organic pollutants with the multidrug resistance transport protein ABCB1 (974.4)

Multidrug resistance (MDR) transporters, from the ATP binding cassette (ABC) superfamily, are thought to act as determinants of chemical accumulation in cells. Despite a long history of research on the interactions of MDR proteins with pharmaceuticals, little is known about the molecular basis of their interactions with environmental compounds such as persistent organic pollutants (POPs). This research is urgently needed to further understanding of substrate recognition by the transporters and to test the hypothesis that pollutant persistence relates to predictable patterns of interaction with the major MDR transporter ABCB1.We use purified recombinant P‐glycoprotein (ABCB1) from mouse and yellowfin tuna to measure pollutant‐transporter interaction kinetics via ATPase stimulation and fluorophore knock‐off experiments. These kinetic will be compared to what is observed with ABCB1 overexpressed in whole cells. Previously analyzed data on “real‐world” pollutant levels in the cosmopolitan and widely consumed fish species of yellowfin tuna (Thunnus albacares) was used to guide the selection of persistent pollutants. The preliminary results of this study show that the majority of the selected persistent pollutants found in tuna are inhibitors of mouse ABCB1 efflux pump. Thereby, isomeric forms of the same pollutant can differ in their IC50 values up to an order of magnitude while reaching inhibition coefficients in the range of the model inhibitor cyclosporine A.These studies aim at determining the conservation of pollutant‐transporter interactions across marine and mammalian ABCB1 transporters. In addition, we want to investigate whether common structural features of ABC transporters and pollutants govern their interactions. These results will shed light on the question of how transporters can direct pollutant movement through the environment and will lead to new avenues for design of more potent pharmaceuticals and safer industrial chemicals.Grant Funding Source: Supported by NIH and the WAITT Foundation

Jadomycins are cytotoxic to ABCB1-, ABCC1-, and ABCG2-overexpressing MCF7 breast cancer cells

Multidrug resistance remains a major obstacle in the effective treatment of metastatic breast cancer. One mechanism by which multidrug resistance is conferred is the decreased intracellular drug accumulation due to the upregulation of the ATP-binding cassette (ABC) transporters. We have previously demonstrated that jadomycins, polyketide-derived natural products produced by Streptomyces venezuelae ISP5230, inhibit the growth of the human breast ductal carcinoma cell lines T47D and MDA-MB-435. To expand our understanding of jadomycin pharmacology, the goal of the present study was to determine whether the function of ABC efflux transporters affects the anticancer activity of jadomycins to MCF7 breast cancer cells. Seven jadomycin analogs (DNV, B, L, SPhG, F, S, and T) effectively reduced the viability of MCF7 control and ABCB1-, ABCC1-, or ABCG2-overexpressing drug-resistant MCF7 breast cancer cells as measured by methyltetrazolium cell viability assays and lactate dehydrogenase cytotoxicity assays. The inhibition of ABCB1, ABCC1, or ABCG2 with verapamil, MK-571, or Ko-143, respectively, did not augment the cytotoxicity of jadomycins DNV, B, L, SPhG, F, S, or T in drug-resistant MCF7 cells. Furthermore, jadomycins B, L, SPhG, F, S, and T did not increase the intracellular accumulation of ABCB1, ABCC1, or ABCG2 fluorescent substrates in HEK-293 cells stably transfected with ABCB1, ABCC1, or ABCG2. We conclude that jadomycins B, L, SPhG, F, S, and T are effective agents in the eradication of MCF7 breast cancer cells grown in culture, and that their cytotoxicities are minimally affected by ABCB1, ABCC1, and ABCG2 efflux transporter function.

First evidence for toxic defense based on the multixenobiotic resistance (MXR) mechanism in Daphnia magna

The water flea Daphnia magna is widely used as test species in ecotoxicological bioassays. So far, there is no information available to which extent ATP binding cassette (ABC) transporter based multixenobiotic resistance (MXR) counteracts adverse chemical effects in this species. This, however, would be important for assessing to which extent the bio-active potential of a compound determined with this species depends on this cellular defense. We here present molecular, functional and toxicological studies that provide first evidence for ABC transporter-based MXR in D. magna. We cloned putatively MXR-related partial abcb1, abcc1/3, abcc4 and abcc5 coding sequences; respective transcripts were constitutively expressed in different D. magna life stages. MXR associated efflux activity was monitored in D. magna using the fluorescent substrate dyes rhodamine 123, rhodamine B and calcein-AM combined with inhibitors of human ABCB1 and/or ABCC transporter activities reversin 205, MK571 and cyclosporin A. With inhibitors present, efflux of dye substrates was reduced in D. magna in a concentration-dependent mode, as indicated by elevated accumulation of the dyes in D. magna tissues. In animals pre-exposed to mercury, pentachlorophenol or dacthal applied as inducers of ABC transporter expression, levels of some ABC transporter transcripts were increased in some cases showing that these genes can be chemically induced. Likewise, pre-exposure of animals to these chemicals decreased dye accumulation in tissue, indicating enhanced MXR transporter activity, likely associated with higher transporter protein levels. Toxicity assays with toxic transporter substrates mitoxantrone and chlorambucil that were applied singly and in combination with inhibitors were performed to study the tolerance role of Abcb1 and Abcc efflux transporters in D. magna. Joint toxicities of about half of the binary combinations of test compounds applied (substrate/inhibitor, substrate/substrate, inhibitor/inhibitor) were greater than joint effects predicted with mixture toxicity models, which can be explained by chemosensitization through MXR efflux transporter interference. Our data indicate the presence of an MXR efflux system in D. magna. It needs to be considered when assessing the bioactive potential of test compounds with this species. Further, chemosensitization may explain joint toxicities of compound mixtures to D. magna that are higher than expected.

Sex differences in cyclosporine pharmacokinetics and ABCB1 gene expression in mononuclear blood cells in African American and Caucasian renal transplant recipients

Cyclosporine exhibits pharmacokinetic and pharmacodynamic variability in renal transplant recipients (RTR) attributed to P-glycoprotein (P-gp), an ABCB1 efflux transporter that influences bioavailability and intracellular distribution. Data on race and sex influences on P-gp in RTR are lacking. We investigated sex and race influences on cyclosporine pharmacokinetics and ABCB1 gene expression in peripheral blood mononuclear cells (PBMC). Fifty-four female and male African American and Caucasian stable RTR receiving cyclosporine and mycophenolic acid completed a 12-hour study. ABCB1 gene expression was assessed in PBMCs pre-dose and 4 hours after cyclosporine. Statistical analysis used mixed effects models on transformed, normalized ABCB1 expression and cyclosporine pharmacokinetics. Sex and race differences were observed for the dose-normalized area under the concentration curve (AUC0-12 /Dose) [P = .0004], apparent clearance [P = .0004] and clearance/body mass index (CL/BMI) [P = .027] with slowest clearance and greatest drug exposure in females. Sex and race differences were found pre-dose and 4 hours for ABCB1 [P < .0001] with females having less expression than males. ABCB1 differences were observed between pre-dose and 4 hours [P = .0009]. Female RTR had slower cyclosporine clearance and lower ABCB1 gene expression in PBMC suggesting reduced efflux activity and greater intracellular drug exposure.

Bipiperidinyl derivatives of 23-hydroxybetulinic acid reverse resistance of HepG2/ADM and MCF-7/ADR cells

Multidrug resistance (MDR) is a major obstacle to successful chemotherapy for cancer; thus, novel MDR reversers are urgently needed. In the present study, we assessed whether two synthetic derivatives of 23-hydroxybetulinic acid, 3,23-O-diacetyl-17-1,4'-bipiperidinyl betulinic amide (DABB) and 3,23-O-dihydroxy-17-1,4'-bipiperidinyl betulinic amide (DHBB), could reverse MDR induced by ATP-binding cassette (ABC) transporters. Using the MTT assay, we found that DABB and DHBB could enhance the cytotoxicities of ABCB1 substrates doxorubicin, vincristine, and paclitaxel in ABCB1-overexpressing HepG2/ADM and MCF-7/ADR cells, whereas that of a non-ABCB1 substrate cisplatin was unaffected. The ABCB1 substrate accumulation and efflux assay showed that DABB and DHBB not only enhanced the retention of doxorubicin and rhodamine123 but also inhibited the efflux of rhodamine123. Further mechanistic studies by reverse transcription PCR, western blot, and ABCB1 ATPase activity assay indicated that DABB and DHBB suppressed ABCB1 ATPase activity, but did not alter mRNA or protein expression of ABCB1. ABCB1 siRNA pretreatment attenuated the reversal effect of DABB and DHBB, indicating that their reversal effects were partially dependent on ABCB1. Docking analysis also implied that DABB and DHBB bind directly to ABCB1 at a site partly overlapped with that of verapamil. Taken together, our findings suggest that two bipiperidinyl derivatives of 23-hydroxybetulinic acid reverse ABCB1-mediated MDR through modulation of ABCB1 ATPase activity, thereby inhibiting its efflux function in both HepG2/ADM and MCF-7/ADR cells. These findings may contribute toward the development of novel MDR reversers using DABB and DHBB as adjuvant anticancer chemotherapy.

ABCB1 polymorphism predicts escitalopram dose needed for remission in major depression

The ATP-binding cassette family of transporter proteins, subfamily B (MDR/TAP), member 1 (ABCB1) (P-glycoprotein) transporter is a key component of the blood–brain barrier. Many antidepressants are subject to ABCB1 efflux. Functional polymorphisms of ABCB1 may influence central nervous system bioavailability of antidepressants subject to efflux. Single-nucleotide polymorphisms (SNPs) at rs1045642 (C3435T) of ABCB1 have been associated with efflux pump efficiency. This may explain part of the interindividual variation in antidepressant dose needed to remit. Individuals (N=113) with DSM-IV (Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition) major depressive disorder (MDD) were treated with escitalopram (ESC) or venlafaxine (VEN) over 8 weeks. The17-item Hamilton Depression Rating Scale was assessed serially, blind to genotype. SNP rs1045642 of ABCB1 along with two SNPs previously reported to be in linkage disequilibrium with it (rs2032582 and rs1128503) were genotyped. Demographic features, clinical features, P450 metabolizer status and 5-HTTLPR (serotonin-transporter-linked promoter region) genotype were controlled for. Carriers of rs1045642 TT needed on average 11 mg of ESC to remit, whereas TC and CC carriers required 24 and 19 mg, respectively (P=0.0001). This equates to a 2.0- (95% confidence interval=1.5–3.4; P<0.001) fold greater ESC dose needed to remit for C carriers compared with TT carriers at rs1045642. Of VEN-treated subjects carrying TT genotype at rs1045642, 73.3% remitted compared with 12.5% for CC genotype (odds ratio=6.69; 95% confidence interval=1.72–25.9, P=0.006). These data suggest that antidepressant dose needed to remit can be predicted by an ABCB1 SNP. This has the potential clinical translation implications for dose selection and remission from MDD.

Dasatinib targets chronic myeloid leukemia-CD34+ progenitors as effectively as it targets mature cells

Dasatinib is effective in most chronic phase chronic myeloid leukemia patients both in first-line therapy and following imatinib failure. While imatinib uptake into CD34(+) cells is low compared to mononuclear cells, few data evaluate how well dasatinib targets primitive CML cells. This study compares intracellular concentration of dasatinib and Bcr-Abl kinase inhibition in CML-CD34(+) progenitors and mononuclear cells induced by dasatinib. The intracellular concentrations of dasatinib were similar between CML-CD34(+) and mononuclear cells (P=0.8). Similarly, there was no significant difference in the degree of dasatinib-mediated Bcr-Abl kinase inhibition. ABCB1 (MDR1) and ABCG2 inhibitors neither increased dasatinib intracellular concentration nor enhanced dasatinib-mediated Bcr-Abl kinase inhibition. In contrast to nilotinib, we show that dasatinib is not an ABCB1 inhibitor. Thus, dasatinib targets CML-CD34(+) progenitors as effectively as it targets mononuclear cells. ABCB1 and ABCG2 efflux pumps do not appear to influence the intracellular dasatinib concentration in CML-CD34(+) progenitors.

Degree of kinase inhibition achieved in vitro by imatinib and nilotinib is decreased by high levels of ABCB1 but not ABCG2

Imatinib and nilotinib interact with ABCB1 and ABCG2. However, whether they are substrates or inhibitors is a source of conjecture. Here, in vitro, Bcr–Abl kinase inhibition was used to elucidate the impact of ABCB1/ABCG2 overexpression on imatinib and nilotinib transport. High levels of ABCB1 protein in K562-Dox cells resulted in a significantly increased 50% inhibitory concentration (IC50) compared with parental K562 cells for imatinib (IC50IM; 9 µM to 19 µM, p = 0.002) and nilotinib (IC50NIL; 345 nM to 620 nM, p = 0.013). This difference was abrogated by ABCB1 inhibitors. However, overexpression of ABCG2 did not significantly increase IC50IM or IC50NIL or significantly decrease IC50 upon ABCG2 inhibition. Inhibition of ABCB1 but not ABCG2 resulted in a substantial increase in intracellular nilotinib when used at 150 nM but no increase when used at 2 µM. Imatinib and nilotinib appear to be transported by ABCB1 but do not interact strongly with ABCG2. Furthermore, ABCB1 efflux of nilotinib may be concentration-dependent with transport occurring at clinically relevant concentrations.

In vitro cytotoxic activity of novel protoflavone analogs-selectivity against a multi-drug resistant cancer cell line

Protoapigenone (PA), a natural flavonoid possessing an unusual p-quinol moiety on its B ring, is a prospective novel anticancer lead compound currently in development, together with WYC0209, a potent synthetic PA analog. Previously, an increased cytotoxicity was found when a 3–4 carbon long aliphatic side-chain was present at C-1' for analogs of PA, while this was not the case when WYC0209 derivatives expressed the same moiety. Fifteen 1'-O-alkyl protoflavone derivatives were synthesized from genkwanin or 4'-hydroxy-6-methylflavone, thirteen of which are new compounds. All compounds, as well as PA, WYC0209 and fourteen of their previously reported analogs were also tested on a multi-drug resistant sub-cell line (MDR) of L5178 mouse T-cell lymphoma and on its parental counterpart (PAR). In general, derivatives expressing a free hydroxyl group at C-1' exerted the strongest activities, while C-1' substituted compounds were found much weaker. Derivatives of 6-methylflavone showed mild, but statistically significant selectivity against the MDR cell line. The results are in agreement with our previous findings for fundamental structure-activity relationships on protoflavones. 6-methylated protoflavones may serve as valuable leads for developing selective compounds against MDR cancer. Identical activity of other derivatives on the PAR and MDR cell lines suggests that cancer cells cannot confer resistance to protoflavones by ABCB1 efflux pump over-expression.

Abcb and Abcc transporter homologs are expressed and active in larvae and adults of zebra mussel and induced by chemical stress

Multixenobiotic resistance (MXR) of aquatic invertebrates has so far been associated with cellular efflux activity mediated by P-glycoprotein (ABCB1) and MRP (multidrug resistance protein; ABCC) type ABC (ATP binding cassette) transporters. Expression and activity of an abcb1/Abcb1 homolog has been shown in eggs and larvae of the zebra mussel Dreissena polymorpha. Here we report identification of a partial cDNA sequence of an abcc/Abcc homolog from zebra mussel that is transcribed and active as a cellular efflux transporter in embryos and gill tissue of adult mussels. Transcript expression levels were comparatively low in eggs and sharply increased after fertilization, then maintaining high expression levels in 1 and 2 dpf (days post fertilization) larvae. MK571, a known inhibitor of mammalian ABCC transporters, blocks efflux of calcein-am in larvae and gill tissue as indicated by elevated calcein fluorescence; this indicates the presence of active Abcc protein in cells of the larvae and gills. Dacthal and mercury used as chemical stressors both induced expression of abcb1 and abcc mRNAs in larvae; accordingly, assays with calcein-am and ABCB1 inhibitor reversin 205 and ABCC inhibitor MK571 indicated enhanced Abcb1 and Abcc efflux activities. Responses to chemicals were different in gills, where abcb1 transcript abundances were enhanced in dacthal and mercury treatments, whereas abcc mRNA was only increased with mercury. Abcb1 and Abcc activities did not in all cases show increases that were according to respective mRNA levels; thus, Abcc activity was significantly higher with dacthal, whereas Abcb1 activity was unchanged with mercury. Our data indicate that abcb1/Abcb1 and abcc/Abcc transporters are expressed and active in larvae and adult stages of zebra mussel. Expression of both genes is induced as cellular stress response, but regulation appears to differ in larvae and tissue of adult stages.

Significant Activity of Ecdysteroids on the Resistance to Doxorubicin in Mammalian Cancer Cells Expressing the Human ABCB1 Transporter

Multidrug resistance (MDR) is a major cause of failure of cancer chemotherapy. Fifty-eight ecdysteroids, herbal analogues of the insect molting hormone and their semisynthetic derivatives, were tested for their activity against L5178 mouse T-cell lymphoma cells (non-MDR) and their subcell line transfected with pHa MDR1/A retrovirus overexpressing the human ABCB1 efflux pump (MDR cell line). The compounds showed very low antiproliferative activities but modulated the efflux of rhodamine 123 mediated by the ABCB1 transporter. Roughly depending on the polarity, mild to strong synergism or antagonism was observed by combining ecdysteroids with doxorubicin, and specific structure-activity relationships were also found. Our results show the effect of ecdysteroids on MDR cancer cells for the first time. Less polar derivatives may serve as valuable leads toward a potent and safe resistance modulator. Biological significance of the resistance-increasing activity of the most abundant phytoecdysteroids including 20-hydroxyecdysone is yet to be clarified.

The Gene Expressions of hOCT1 and ABCB1 Change During the Course of CML in Relation to Imatinib Therapy

Abstract 2495 Background.The intracellular concentration of imatinib (IM) in patients with chronic myeloid leukemia (CML) is supposed to be influenced by the expression of its main cellular transporters, hOCT1 (influx) and ABCB1 (efflux). The assessment of the genes expression may be potentially important in clinical practice to effectively manage the therapy. Our recent results showed the necessity to cautiously interpret results from gene expression measurements of both transporters as their detected mRNA levels are affected by the proportion of different cell types in the sample analyzed (Racil et al. 2010 Am J Hem 85:525, Racil et al. 2011 Leuk & Lymph 52:331). Aims. In this report, we aimed to comprehensively assess differences in the expression of hOCT1 and ABCB1 in total leukocytes of peripheral blood (PB) in relation to the blood cell lineage in CML patients with different responses to IM. Methods. Kruskall Wallis’s and Dunn’s multiple comparison tests were applied to calculate differences in transcript levels of hOCT1 and ABCB1 in patients at diagnosis (Dg=43), in major molecular response (MMR=27), complete molecular response (BCR-ABL log 4.5 reduction; CMR4.5 =15), therapy failure (TF=13), accelerated phase (AP=12) and in 75 healthy controls. CMR4.5 is defined here as either a detectable disease ≤0.0032% BCR-ABLIS or as undetectable by nested PCR. TF is defined as non CCgR achievement. Additionally, we used the Spearman’s correlation test to investigate relationship between expressions and percentage of immature cells and neutrophils in patients with non-physiological blood count (Dg and AP). In patients with normal blood count (CMR4.5, MMR, TF), we calculated correlation with BCR-ABL transcript level. Finally, we performed in vitro experiments with BCR-ABL negative (SKM-1, MOLM-13) and positive cell lines (K562, MOLM-7) treated with IM to study its effect on ABCB1 expression. Results. We found a significantly lower expression of hOCT1 and ABCB1 at Dg (P<0.001) and in AP (P<0.001 and P<0.01, respectively) in comparison to controls. The lower expression probably depends indirectly on the immature cells count (hOCT1 r = −0.4850, P=0.0002; ABCB1 r = −0.6451, P< 0.0001). Interestingly, while a significant positive correlation of neutrophil count was found with hOCT1 mRNA levels (r=0.6090, P< 0.0001), no correlation was observed with ABCB1. In patients with physiological blood count, we observed significantly elevated expression of ABCB1 in MMR and CMR4.5 in comparison to controls (P< 0.01). As MMR and CMR4.5 samples are characterized by the BCR-ABL transcript level <0.1%, this result led us to perform in vitro experiments to test the effect of IM on ABCB1 expression in BCR-ABL negative cell lines showing an elevated expression after incubation with IM. We assume this effect to be a natural defense of BCR-ABL negative cells to get rid of IM. By contrast, we found a drop in ABCB1 expression after BCR-ABL positive cell lines incubation with IM, suggesting inhibitory effects of IM. A significantly reduced expression of hOCT1 was found in TF in comparison to MMR and CMR4.5 (P< 0.01). These samples did not differ in blood count, but we observed a significant negative correlation of hOCT1 with BCR-ABL transcript levels (r= − 0.5117; P< 0.0001). Conclusion. Cell composition in PB changes during the CML treatment with IM which influences the measurement of hOCT1 and ABCB1 mRNA levels in total leukocytes. However, hOCT1 expression was found to be significantly lower in patients who failed to achieve CCgR but had normal blood count. Interestingly, our data suggest that ABCB1 is differently expressed in BCR-ABL positive and negative cells due to the inhibitory and enhancing effects of IM, respectively. Supported by IGA NT11555 and MZOUHKT2005 Disclosures:Machova Polakova:Novartis: travel grant. Ondrackova:BMS: travel grant.

Alkoxy-auxins Are Selective Inhibitors of Auxin Transport Mediated by PIN, ABCB, and AUX1 Transporters

Polar auxin movement is a primary regulator of programmed and plastic plant development. Auxin transport is highly regulated at the cellular level and is mediated by coordinated transport activity of plasma membrane-localized PIN, ABCB, and AUX1/LAX transporters. The activity of these transporters has been extensively analyzed using a combination of pharmacological inhibitors, synthetic auxins, and knock-out mutants in Arabidopsis. However, efforts to analyze auxin-dependent growth in other species that are less tractable to genetic manipulation require more selective inhibitors than are currently available. In this report, we characterize the inhibitory activity of 5-alkoxy derivatives of indole 3-acetic acid and 7-alkoxy derivatives of naphthalene 1-acetic acid, finding that the hexyloxy and benzyloxy derivatives act as potent inhibitors of auxin action in plants. These alkoxy-auxin analogs inhibit polar auxin transport and tropic responses associated with asymmetric auxin distribution in Arabidopsis and maize. The alkoxy-auxin analogs inhibit auxin transport mediated by AUX1, PIN, and ABCB proteins expressed in yeast. However, these analogs did not inhibit or activate SCF(TIR1) auxin signaling and had no effect on the subcellular trafficking of PIN proteins. Together these results indicate that alkoxy-auxins are inactive auxin analogs for auxin signaling, but are recognized by PIN, ABCB, and AUX1 auxin transport proteins. Alkoxy-auxins are powerful new tools for analyses of auxin-dependent development.

Total Synthesis of Natural and Non-Natural Δ5,6Δ12,13-Jatrophane Diterpenes and Their Evaluation as MDR Modulators

We report the details of the total synthesis of natural and non-natural jatropha-5,12-dienes. The successful tactic for the assembly of the strained trans-bicyclo[10.3.0]pentadecane scaffold employed a B-alkyl Suzuki-Miyaura cross-coupling for the formation of the C5/C6 double bond and a ring-closing metathesis for the construction of the C12/C13 double bond. The key step of the synthesis of the cyclopentane fragment, an uncatalyzed intramolecular carbonyl-ene reaction, was studied computationally by DFT calculations. The members of the ensemble of synthetic natural and non-natural jatrophanes were subsequently examined as modulators for the ABCB1, ABCG2, and ABCC1 efflux proteins, which are associated with multidrug resistance in cancer chemotherapy.

Spathulenol inhibit the human ABCB1 efflux pump

Since multidrug resistance (MDR) is a major cause of failure in cancer chemotherapy, the search for new compounds that can be used as adjuvants of chemotherapy is urgent. This study focuses on the evaluation of cytotoxicity and MDR reversal activity of eleven compounds representing diverse structural types of Asteraceae sesquiterpenes. Xanthatin, 4-epixanthanol, sintenin, cnicin, 4′-acetylcnicin, 3b-hydroxycostunolide, desacetylmatricarin, paulitin, isoalantolactone, chrysanin and spathulenol were tested, by flow cytometry, for their activities as modulators of the efflux of rhodamine123 by the human ABCB1 (commonly known as P-gp) pump. Two cell lines were used: a L5178 mouse T-cell lymphoma cell line (PAR cell line) and the L5178 mouse T-cell lymphoma cells transfected with pHa MDR1/A retrovirus (MDR cell line). It was observed (figure 1) that spathulenol highly promoted the accumulation of rhodamine123 (substrate of the ABCB1) by the MDR cells, which over-expresses the ABCB1 efflux pump. Spathulenol, sintenin and desacetylmatricarin presented moderate or low cytotoxicity with IC50 higher than 6µM, while the remaining compounds presented higher cytotoxicity against the two cell lines tested. The results with spathulenol suggest that this compound is a good candidate to be used in combination chemotherapy of MDR cancer and therefore is worthy for further in vivo studies.

Blocking of Cytokine Survival Signals along with Intense Bcr-Abl Kinase Inhibition May Eradicate CML Progenitor Cells.

Abstract 3250Poster Board III-1▪▪This icon denotes an abstract that is clinically relevant.Preclinical studies of imatinib set the paradigm of continuous Bcr-Abl kinase inhibition for optimal response in chronic myeloid leukemia (CML). However, the clinical success of once daily dasatinib, despite its short serum half life, implies that intermittent inhibition of Bcr-Abl kinase activity is sufficient for clinical response. In vitro studies also demonstrated that short-term intense (≥90%) Bcr-Abl kinase inhibition triggers cell death in BCR-ABL + cell lines, demonstrating their oncogene addiction. However, the effect of short-term intense kinase inhibition on CD34+ CML progenitors is not studied. Clinical, mathematical modelling and in vitro studies suggest that leukemic stem cells (LSC) are difficult to eradicate and hence the majority of CML patients may not be cured with tyrosine kinase inhibitors (TKI). Inadequate Bcr-Abl kinase inhibition has been postulated to cause refractoriness of LSC to TKI's. This may be due to increased expression of ABCB1 and ABCG2 efflux proteins, or the quiescent state of LSC. However, the phenomenon could be independent of Bcr-Abl kinase activity. In vivo leukemic progenitors live in a cytokine rich environment which may be providing a mechanism for Bcr-Abl independent resistance. We have assessed the impact of short-term intense Bcr-Abl kinase inhibition on CML cell lines and CML CD34+ primary cells in the presence and absence of cytokines. In CML cell lines, short-term (cells were cultured with dasatinib for 30 min and following thorough drug washout, cells were recultured in drug free media for 72 hr) intense Bcr-Abl kinase inhibition with 100 nM dasatinib triggers cell death. In CML-CD34+ cells 30 min of culture with 100 nM dasatinib (n=13) or 30 μM IM (n=7) reduced the level of p-Crkl (surrogate marker of Bcr-Abl kinase activity) by 97±3% and 96±4% respectively. In the presence of either a six growth factors cocktail (6-GF; n=10) or GM-CSF (n=11) or G-CSF (n=4) alone, despite 97% inhibition of p-Crkl, short-term culture with 100 nM dasatinib (D100ST) reduced colony forming cells (CFC) by only 24%, 32% or 5%, respectively. However without cytokines, D100ST reduced CML-CD34+ CFCs by 70%. Consistent with the results observed with dasatinib, short-term culture with 30 μM imatinib (IM) (n=3) also reduced 90% CFC in the absence of cytokines but by only 38% in the presence of 6-GF. These results suggest that in CML-CD34+ cells, GM-CSF, G-CSF or 6-GF mediate Bcr-Abl independent TKI resistance. It is possible that cytokines may be promoting cell survival via signalling pathways that are refractory to dasatinib. To examine this possibility, we assessed the effect of D100ST on p-STAT5 signalling in CML-CD34+ cells, in the presence and absence of GM-CSF, G-CSF or 6-GF. STAT5 was constitutively phosphorylated in CML-CD34+ cells, and in the absence of cytokines, D100ST reduced the p-STAT 5. STAT5 phosphorylation was not inhibited by D100ST when cells were cultured with 6-GFs or GM-CSF however, the combination of D100ST and a Janus kinase (Jak) inhibitor dramatically reduced p-STAT5. Similarly, in the presence of GM-CSF (32.35±5.16% vs. 68.33±14.90%) or G-CSF (58.13±13 vs. 94.68±21.12) combination of D100ST and JAK inhibitor significantly reduced CFC compared to D100ST only. Thus our data suggest that in contrast to CML cell lines, primary CML progenitors may not be completely dependent on the BCR-ABL oncogene and that activation of the cytokine mediated JAK-2/STAT-5 pathway may circumvent the need for BCR-ABL signalling for maintenance of survival. Thus a therapeutic strategy based on short-term intense kinase inhibition may have limited success unless critical redundant cytokine-induced survival pathways are also inhibited. We postulate that blockade of cytokine signalling along with short-term intense Bcr-Abl kinase inhibition with a potent second generation TKI may provide a novel strategy to eradicate primitive CML cells. [Display omitted] Disclosures:White:Novartis and Britol-Myers Squibb: Research Funding. Hughes:BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

A Novel Two Mode-Acting Inhibitor of ABCG2-Mediated Multidrug Transport and Resistance in Cancer Chemotherapy

BackgroundMultidrug resistance (MDR) is a major problem in successful treatment of cancers. Human ABCG2, a member of the ATP-binding cassette transporter superfamily, plays a key role in MDR and an important role in protecting cancer stem cells. Knockout of ABCG2 had no apparent adverse effect on the mice. Thus, ABCG2 is an ideal target for development of chemo-sensitizing agents for better treatment of drug resistant cancers and helping eradicate cancer stem cells.Methods/Preliminary FindingsUsing rational screening of representatives from a chemical compound library, we found a novel inhibitor of ABCG2, PZ-39 (N-(4-chlorophenyl)-2-[(6-{[4,6-di(4-morpholinyl)-1,3,5-triazin-2-yl]amino}-1,3-benzothiazol-2-yl)sulfanyl]acetamide), that has two modes of actions by inhibiting ABCG2 activity and by accelerating its lysosome-dependent degradation. PZ-39 has no effect on ABCB1 and ABCC1-mediated drug efflux, resistance, and their expression, indicating that it may be specific to ABCG2. Analyses of its analogue compounds showed that the pharmacophore of PZ-39 is benzothiazole linked to a triazine ring backbone.Conclusion/SignificanceUnlike any previously known ABCG2 transporter inhibitors, PZ-39 has a novel two-mode action by inhibiting ABCG2 activity, an acute effect, and by accelerating lysosome-dependent degradation, a chronic effect. PZ-39 is potentially a valuable probe for structure-function studies of ABCG2 and a lead compound for developing therapeutics targeting ABCG2-mediated MDR in combinational cancer chemotherapy.

Novel E-ring camptothecin keto analogues (S38809 and S39625) are stable, potent, and selective topoisomerase I inhibitors without being substrates of drug efflux transporters

Camptothecin (CPT) analogues are powerful anticancer agents but are chemically unstable due to their alpha-hydroxylactone six-membered E-ring structure, which is essential for trapping topoisomerase I (Top1)-DNA cleavage complexes. To stabilize the E-ring, CPT keto analogues with a five-membered E-ring lacking the oxygen of the lactone ring (S38809 and S39625) have been synthesized. S39625 has been selected for advanced preclinical development based on its promising activity in tumor models. Here, we show that both keto analogues are active against purified Top1 and selective against Top1 in yeast and human cancer cells. The keto analogues show improved cytotoxicity toward colon, breast, and prostate cancer cells and leukemia cells compared with CPT. The drug-induced Top1-DNA cleavage complexes induced by the keto analogues show remarkable persistence both with purified Top1 and in cells following 1-h drug treatments. Moreover, we find that S39625 is not a substrate for either the ABCB1 (multidrug resistance-1/P-glycoprotein) or ABCG2 (mitoxantrone resistance/breast cancer resistance protein) drug efflux transporters, which sets S39625 apart from the clinically used CPT analogues topotecan or SN-38 (active metabolite of irinotecan). Finally, we show that nanomolar concentrations of S38809 or S39625 induce intense and persistent histone gamma-H2AX. The chemical stability of the keto analogues and the ability of S39625 to produce high levels of persistent Top1-DNA cleavage complex and its potent antiproliferative activity against human cancer cell lines make S39625 a promising new anticancer drug candidate. Histone gamma-H2AX could be used as a biomarker for the upcoming clinical trials of S39625.