Drug-resistant KB Research Articles

Reversal of P-glycoprotein-mediated multidrug resistance by natural N-alkylated indole alkaloid derivatives in KB-ChR-8-5 drug-resistant cancer cells.

Multidrug resistance (MDR) remains a significant challenge in cancer chemotherapy due to the overexpression of ATP-binding cassette drug-efflux transporters, namely P-glycoprotein (P-gp)/ATP-binding cassette subfamily B member 1. In this study, derivatives of N-alkylated monoterpene indole alkaloids such as N-(para-bromobenzyl) (NBBT), N-(para-methylbenzyl) (NMBT), and N-(para-methoxyphenethyl) (NMPT)moieties were investigated for the reversal of P-gp-mediated MDRin drug-resistant KB colchicine-resistant 8-5(KB-ChR-8-5) cells. Among the three indole alkaloid derivatives, the NBBT exhibited the highest P-gp inhibitory activity in a dose-dependent manner. Further, it significantly decreased P-gp overexpression by inactivating the nuclear translocation of the nuclearfactor kappa B p-50 subunit. In the cell survival assay, doxorubicin showed 6.3-fold resistance (FR) in KB-ChR-8-5 cells compared with its parental KB-3-1 cells. However, NBBT significantly reduced doxorubicin FR to 1.7, 1.3, and 0.4 and showed strong synergism with doxorubicin for all the concentrations studied in the drug-resistant cells. Furthermore, NBBT and doxorubicin combination decreased the cellular migration and showed increased apoptotic incidence by downregulating Bcl-2, then activating BAX, caspase 3, and p53. The present findings suggest that NBBT could be a lead candidate for the reversal of P-gp- mediated multidrug resistance in cancer cells.

PAMAM G4.5 dendrimers for targeted delivery of ferulic acid and paclitaxel to overcome P-glycoprotein-mediated multidrug resistance.

In this study, we prepared ferulic acid (FA) and paclitaxel (PTX) co-loaded polyamidoamine (PAMAM) dendrimers conjugated with arginyl-glycyl-aspartic acid (RGD) to overcome P-glycoprotein (P-gp)-mediated multidrug resistance (MDR). FA was released in greater extent (80%) from the outer layer of the dendrimers compared with PTX (70%) from the interior of the dendrimers. FA improved intracellular availability of PTX via P-gp modulation in drug-resistant cells. In vitro drug uptake data show higher PTX delivery with RGD-PAMAM-FP than with PAMAM-FP in drug resistant KB CH-R 8-5 cell lines. This indicates that RGD facilitates intracellular PTX accumulation through active targeting in multidrug-resistant KB CH-R 8-5 cells. The terminal deoxynucleotidyl transferase 2'-deoxyuridine 5'-triphosphate nick-end labeling assay data and membrane potential analysis in mitochondria confirm the enhanced anticancer potential of RGD-PAMAM-FP nanoaggregates in drug-resistant cells. We also confirmed by the increased protein levels of proapoptotic factors such as caspase 3, caspase 9, p53, and Bax after treatment with RGD-PAMAM-FP nanoaggregates and also downregulates antiapoptotic factors. Hence, FA-PTX co-loaded, RGD-functionalized PAMAM G4.5 dendrimers may be considered as an effective therapeutic strategy to induce apoptosis in P-gp-overexpressing, multidrug-resistant cells.

Ferulic acid reverses ABCB1-mediated paclitaxel resistance in MDR cell lines

Multidrug resistance (MDR) remains a major obstacle in cancer chemotherapy. The use of the dietary phytochemicals as chemosensitizing agents to enhance the efficacy of conventional cytostatic drugs has recently gained the attention as a plausible approach for overcoming the drug resistance. The aim of this study was to investigate whether a naturally occurring diet-based phenolic acid, ferulic acid, could sensitize paclitaxel efficacy in ABCB1 overexpressing (P-glycoprotein) colchicine selected KB ChR8-5 cell line. In vitro drug efflux assays demonstrated that ferulic acid inhibits P-glycoprotein transport function in drug resistant KB ChR8-5 cell lines. However, ferulic acid significantly downregulates ABCB1 expression in a concentration dependent manner. Cytotoxicity assay reveals that ferulic acid decreased paclitaxel resistance in KBChR8-5 and HEK293/ABCB1 cells, which indicates its chemosensitizing potential. Clonogenic cell survival assay and apoptotic morphological staining further confirm the chemosensitizing potential of ferulic acid in drug resistant KB ChR8-5 cell lines. Ferulic acid treatment enhances paclitaxel mediated cell cycle arrest and upregulates paclitaxel-induced apoptotic signaling in KB resistant cells. Hence, it has been concluded that downregulation of ABCB1 and subsequent induction of paclitaxel-mediated cell cycle arrest and apoptotic signaling may be the cause for the chemosensitizing potential of ferulic acid in P-gp overexpressing cell lines.

Macrodasines A–G, macroline indole alkaloids incorporating fused spirocyclic tetrahydrofuran–tetrahydrofuran and tetrahydrofuran–tetrahydropyran rings

The bark extract of the Malayan Alstonia angustifolia Wall provided the spirocyclic alkaloids macrodasines A–G. The structures of the new compounds were established by analysis of the spectroscopic data and in the case of macrodasines A and B confirmed by X-ray diffraction analysis. Macrodasines A, B, C, and G incorporate fused spirocyclic tetrahydrofuran–tetrahydrofuran rings, while macrodasines D, E, and F incorporate fused tetrahydrofuran–tetrahydropyran rings. Macrodasines B, C, and E were found to show moderate levels of activity in reversing multidrug-resistance in drug-resistant KB cells.

Synthesis, Characterization, and Molecular Structure of a Novel Zinc (II) Complex: Assessment of Impact of MDR1Pgp Expression on its Cytotoxic Activity

Zinc(II)complex (3) {bis(3-ethoxy-2-hydroxy-benzylidene)-N,N'-bis(2,2-dimethyl-3-aminopropyl)ethylenediamine}-zinc(II); [(3-OEt-ENBDMPI)Zn(II)] was obtained in situ by a ligand exchange reaction involving zinc(II) acetylacetonate and the Schiff-base ligand obtained in situ. For assessing ability of 3 to act as a transport substrate of multidrug resistance (MDR1) P-glycoprotein (Pgp), its cytotoxic activity was evaluated in human epidermal carcinoma drug-sensitive KB 3-1 (Pgp-) and drug resistant KB 8-5 (Pgp+) cells. Compared with its cationic gallium(III) counterpart 4 showing cytotoxicity profiles consistent with its recognition as a Pgp substrate, the neutral zinc(II) complex 3 did not display cytotoxicity profiles (at pharmacologically relevant concentrations <10 µM) modified by expression of Pgp. Further, 3 was found be slightly more toxic against KB 8-5 cells compared to KB 3-1 cells at higher concentration. The neutral zinc (II) complex 3 was also found to be considerably less toxic against Pgp-lacking cells compared to its cationic gallium(III) counterpart 4. Additionally, the neutral zinc(II) complex 3 demonstrated considerably more toxicity against Pgp expressing KB 8-5 cells (> 10 µM) compared with its cationic counterpart 4 displaying minimal effect at highest concentration. The results suggest that differential cytotoxic activity of 3 and 4 in drug-resistant human epidermal carcinoma KB 8-5 (Pgp+) cells could result from variation in the overall charge of the molecules.

Impaired p53 binding to importin: a novel mechanism of cytoplasmic sequestration identified in oxaliplatin-resistant cells

Previous studies have described one nuclear localization signal (NLSI) in p53 and speculated on two additional sites termed NLSII and NLSIII. Drug-resistant KB cells selected with cisplatin or oxaliplatin were found to have increased p53 levels and in oxaliplatin-selected cells, a larger p53 predominantly in the cytoplasm. In oxaliplatin-selected cells a single nucleotide deletion in the sequence-encoding amino acid 382, part of NLSIII, resulted in a frame shift and a 420 amino acid protein (p53(420)). We investigated explanations for the cytoplasmic sequestration of p53(420) while assessing the role, if any, of NLSII and NLSIII in p53 nuclear import. We found that neither NLSII nor NLSIII are essential for p53 nuclear localization. Furthermore, we confirmed p53(420) is able to tetramerize, transactivate a p21 promoter, bind dynein and that the reduced nuclear accumulation is not a consequence of increased p53 nuclear export. However, the association of p53(420) with importin-beta, essential for nuclear import, was significantly impaired. We conclude that despite sequence similarity to consensus NLSs neither NLSII nor NLSIII have roles in p53 nuclear transport. We also identified impaired association with importin as a novel mechanism of p53 cytoplasmic sequestration that impairs nuclear transport rendering cells functionally deficient in p53.

The alkaloids of the mersinine group: a new subclass of the monoterpenoid indole alkaloids from Kopsia

A total of 16 alkaloids, exemplified by mersinine A and its congeners, and constituting a new subclass of the monoterpenoid indoles, were isolated exclusively and for the first time from Kopsia singapurensis. The structures of these alkaloids were established by spectroscopic methods and in some instances confirmed by X-ray diffraction analysis. A possible biogenetic route from an aspidofractinine precursor is proposed. Compounds 1, 2, 3, 9, and 10 were found to reverse multidrug-resistance in drug-resistant KB cells.

Biologically Active Aspidofractinine, Rhazinilam, Akuammiline, and Vincorine Alkaloids from Kopsia

Eleven new indole alkaloids, in addition to the previously reported rhazinal (1), and 14 other known alkaloids, were obtained from the Malayan Kopsia singapurensis, viz., kopsiloscines A-F (2-7), 16-epikopsinine (8), kopsilongine- N-oxide (9), 16-epiakuammiline (10), aspidophylline A (11), and vincophylline (12). The structures of these alkaloids were determined using NMR and MS analyses. Rhazinal (1), rhazinilam (17), and rhazinicine (18) showed appreciable cytotoxicity toward drug-sensitive as well as vincristine-resistant KB cells, while kopsiloscines A (2), B (3), and D (5) and aspidophylline A (11) were found to reverse drug-resistance in drug-resistant KB cells.

Lundurines A—D, Cytotoxic Indole Alkaloids Incorporating a Cyclopropyl Moiety from Kopsia tenuis and Revision of the Structures of Tenuisines A—C.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Lundurines A–D, cytotoxic indole alkaloids incorporating a cyclopropyl moiety from Kopsia tenuis and revision of the structures of tenuisines A–C

The leaf extract of the Borneo Kopsia, K. tenuis, provided several new indoles with a novel hexacyclic carbon skeleton, incorporating a cyclopropyl moiety. Two of these, displayed appreciable in vitro cytotoxicity against B16 melanoma cells, as well as the circumvention of drug-resistance in drug-resistant KB cells. The structures of tenuisines A–C were revised from a dimeric to a monomeric structure, based on new LSIMS data, and in conjunction with the preparation of the methyl iodide salt of tenuisine A.

Characterization of a novel 99mTc-carbonyl complex as a functional probe of MDR1 P-glycoprotein transport activity.

Multidrug resistance (MDR) mediated by overexpression of MDR1 P-glycoprotein (Pgp) is one of the best characterized barriers to chemotherapy in cancer patients. Furthermore, the protective function of Pgp-mediated efflux of xenobiotics in various organs has a profound effect on the bioavailability of drugs in general. Thus, there is an expanding requirement to noninvasively interrogate Pgp transport activity in vivo. We herein report the Pgp recognition properties of a novel 99mTc(I)-tricarbonyl complex, [99mTc(CO)3(MIBI)3]+ (Tc-CO-MIBI). Tc-CO-MIBI showed 60-fold higher accumulation in drug-sensitive KB 3-1 cells compared to colchicine-selected drug-resistant KB 8-5 cells. In KB 8-5 cells, tracer enhancement was observed with the potent MDR modulator LY335979 (EC50 = 62 nM). Similar behavior was observed using drug-sensitive MCF-7 breast adenocarcinoma cells and MCF-7/MDR1 stable transfectants, confirming that Tc-CO-MIBI is specifically excluded by overexpression of MDR1 Pgp. By comparison, net accumulation in control H69 lung tumor cells was 9-fold higher than in MDR-associated protein (MRP1)-expressing H69AR cells, indicating only modest transport by MRP1. Biodistribution analysis following tail vein injection of Tc-CO-MIBI showed delayed liver clearance as well as enhanced brain uptake and retention in mdr1a/1b(-/-) gene deleted mice versus wild-type mice, directly demonstrating that Tc-CO-MIBI is a functional probe of Pgp transport activity in vivo.

Characterization of a Novel 99mTc-Carbonyl Complex as a Functional Probe of MDR1 P-Glycoprotein Transport Activity

Multidrug resistance (MDR) mediated by overexpression of MDR1 P-glycoprotein (Pgp) is one of the best characterized barriers to chemotherapy in cancer patients. Furthermore, the protective function of Pgp-mediated efflux of xenobiotics in various organs has a profound effect on the bioavailability of drugs in general. Thus, there is an expanding requirement to noninvasively interrogate Pgp transport activity in vivo. We herein report the Pgp recognition properties of a novel 99mTc(I)-tricarbonyl complex, [99mTc(CO)3(MIBI)3]+ (Tc-CO-MIBI). Tc-CO-MIBI showed 60-fold higher accumulation in drug-sensitive KB 3-1 cells compared to colchicine-selected drug-resistant KB 8-5 cells. In KB 8-5 cells, tracer enhancement was observed with the potent MDR modulator LY335979 (EC50 = 62 nM). Similar behavior was observed using drug-sensitive MCF-7 breast adenocarcinoma cells and MCF-7/MDR1 stable transfectants, confirming that Tc-CO-MIBI is specifically excluded by overexpression of MDR1 Pgp. By comparison, net accumulation in control H69 lung tumor cells was 9-fold higher than in MDR-associated protein (MRP1)-expressing H69AR cells, indicating only modest transport by MRP1. Biodistribution analysis following tail vein injection of Tc-CO-MIBI showed delayed liver clearance as well as enhanced brain uptake and retention in mdr1a/1b(-/-) gene deleted mice versus wild-type mice, directly demonstrating that Tc-CO-MIBI is a functional probe of Pgp transport activity in vivo.

Antitumor agents 201. 1 Cytotoxicity of harmine and β-carboline analogs

Twenty-six β-carbolines were evaluated for in vitro cytotoxicity in a human tumor cell line panel. Harmine ( 3) showed significant activity against several cell lines including three drug-resistant KB sublines with various resistance mechanisms. α-(4-Nitrobenzylidine) harmine ( 16) had a broad cytotoxicity spectrum (ED 50 values from 0.3–1.2 μg/mL against 1A9, KB, SaOS-2, A549, SK-MEL-2, U-87-MG, and MCF-7 cells).

Reversal of multidrug resistance by kopsiflorine isolated from Kopsia dasyrachis.

Kopsiflorine, an indole alkaloid of the aspidofractinine-type isolated from Kopsia dasyrachis, was examined for its effect in enhancing drug cytotoxicity in multidrug-resistant tumor cells. The cytotoxicity of vincristine was enhanced in a concentration-dependent manner by kopsiflorine in drug-resistant KB cells (VJ-300). Kopsiflorine alone had no effect on the growth of drug sensitive or resistant cells, but the intracellular accumulation of vincristine was enhanced by kopsiflorine in VJ-300 cells. Kopsiflorine (10 micrograms/ml) significantly inhibited the binding of [3H]azidopine to P-glycoprotein in VJ-300 cells. The results suggest that kopsiflorine interacts directly with P-glycoprotein and inhibits the efflux of antitumor agents in drug-resistant cells.

Antitumor Agents. 192. Antitubulin Effect and Cytotoxicity of C(7)-Oxygenated Allocolchicinoids

Two allocolchicinoids 6 and 8, prepared from colchicine, together with allo compounds 9-11, made from 6 by reduction and regiodemethylation, were evaluated for antitubulin and antitumor activities. Structures of 6, 8, and 10 were confirmed by X-ray crystallographic analysis. Compounds 6, 8, and 9 have high tubulin binding affinity and display potent inhibitory activities against tubulin polymerization and solid human tumor cell lines. Particularly, drug-resistant KB cell lines, including KB-7d, KB-VCR, and KB-CPT, do not show marked resistance to these compounds.

Antitumor agents—CLXXIII. Synthesis and evaluation of camptothecin-4β-amino-4′- O-demethyl epipodophyllotoxin conjugates as inhibitors of mammalian DNA topoisomerases and as cytotoxic agents

Two conjugates composed of a camptothecin and a 4′- O-demethyl epipodophyllotoxin derivative joined by an imine linkage were prepared and evaluated as inhibitors of mammalian DNA topoisomerases I and II. Target compounds stimulated cleavable complex formation with both types of enzyme in vitro although activities were reduced at least twofold relative to the activity of unconjugated constituents. The behavior of the most active conjugate as an inhibitor of cell growth closely resembled both topoisomerase I- and II- inhibitory components in that the compound displayed a combined spectrum of activity against various drug-resistant KB sublines. Cytotoxic activity and selectivity were largely retained through conjugation, the exception being a lower than expected activity against a pleiotrophic multidrug-resistant subline. The induced levels and the properties of cellular protein-associated DNA complexes were consistent with topoisomerase involvement and with the in vitro cleavage assay results. Based on the present findings, conjugation afforded cleavable complex-forming topoisomerase inhibitors which display dual target specificity and a broad spectrum of cytotoxic activity against drug-resistant cells.

Agents which reverse multidrug-resistance are inhibitors of [ 3H]vinblastine transport by isolated vesicles

Resistance of human cancer cells to multiple cytotoxic hydrophobic agents (multidrug resistance) is due to overexpression of the MDR1 gene whose product is the ATP-dependent multidrug transporter, P-glycoprotein. We have previously reported that plasma membrane vesicles partially purified from multidrug-resistant human KB carcinoma cells, but not from drug-sensitive cells, accumulated [ 3H]vinblastine in an ATP-dependent manner (Horio, M., Gottesman, M.M. and Pastan, I. (1988) Proc. Natl. Acad. Sci. USA 85, 3580–3584). Certain calcium-channel blockers, quinidine, and phenothiazines are able to overcome multidrug resistance in cultured cells. In this work, the effect of these reversing agents on ATP-dependent vinblastine (VBL) transport by vesicles from drug-resistant KB cells has been characterized. Azidopine was the most potent inhibitor of ATP-dependent VBL uptake tested (ID 50; concentration of inhibitor such that the transport of vinblastine is inhibited by 50%, < 1 μM). Verapamil, quinidine, and the tiapamil analogue RO-11-2933 were potent but less effective inhibitors (ID 50 < 5 μM). Diltiazem, nifedipine and trifluoperazine were even less effective. These agents had no effect on Na +-dependent and Na +-independent l-leucine uptake by the vesicles, indicating that the inhibition of ATP dependent VBL transport by these agents is not a non-specific effect, as might result from leaks in the vesicle membrane. Verapamil, quinidine, azidopine and trifluoperazine increased the apparent K m value of vinblastine transport, suggesting that these agents may be competitive inhibitors of vinblastine transport.

Stability and covalent modification of P-glycoprotein in multidrug-resistant KB cells.

An antipeptide antibody (P7) to P-glycoprotein has been produced by immunizing rabbits with a synthetic peptide. Antibody P7 is directed against the amino-terminal region of P170 (residues 28-35). The antibody immunoprecipitates a 170-kDa P-glycoprotein from extracts of drug-resistant KB-V1 cells that is not present in the drug-sensitive cell line KB-3-1. Antibody P7 was used to quantitate the amount of P-glycoprotein present in drug-resistant KB lines at various levels of resistance and to demonstrate the presence of P-glycoprotein in NIH 3T3 cells transfected with a cloned MDR1 cDNA or human genomic DNA encoding MDR1. Pulse-chase labeling experiments demonstrated that P-glycoprotein is synthesized as a 140-kDa precursor which is slowly converted over 2-4 h to a 170-kDa glycoprotein. Tunicamycin treatment blocks the conversion of the precursor to the mature form, and removal of N-linked oligosaccharides with Endo F reduces the relative molecular weight of P-glycoprotein to 140K. The mobility of mature P-glycoprotein is unaffected by treatment with neuraminidase and Endo H. These data indicate that P-glycoprotein is N-glycosylated and contains little or no neuraminic acid. P-Glycoprotein is also phosphorylated, and the extent of phosphate incorporated is proportional to the amount of protein present in drug-resistant cells.